Thermo Fisher Scientific NanoDrop One User Manual

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NanoDrop Micro-UV/Vis Spectrophotometer

NanoDrop One

User Guide

269-309102 NanoDrop One UG Revision C March 2021

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Wi-Fi is either a trademark or a registered trademark of Wi-Fi Alliance in the United States and/or other countries. Bluetooth is either a trademark or a registered trademark of Bluetooth Special Interest Group. Windows is either a trademark or a registered trademark of Microsoft Corporation in the United States and/or other countries. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.

For U.S. Technical Support, please contact:

Thermo Fisher Scientific 3411 Silverside Road Tatnall Building, Suite 100 Wilmington, DE 19810 U.S.A.

Telephone: 302 479 7707 Toll Free: 1 877 724 7690 (U.S. & Canada only)

For International Support, please contact:

http://www.thermofisher.com/ NanoDropSupport

Contact your local distributor. For contact information go to:

http://www.thermofisher.com/ NanoDropDistributors

E-mail: nanodrop@thermofisher.com

Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the product operation. This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc.

The contents of this document are subject to change without notice. All technical information in this document is for reference purposes only. System configurations and specifications in this document supersede all previous information received by the purchaser.

Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or error-free and assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might result from any use of this document, even if the information in the document is followed properly.

This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale shall govern all conflicting information between the two documents.

For Research Use Only. This instrument or accessory is not a medical device and is not intended to be used for the prevention, diagnosis, treatment or cure of disease.

WARNING Avoid an explosion or fire hazard. This instrument or accessory is not designed for use in an explosive atmosphere.

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Chapter 1 About the Spectrophotometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

Cuvette Holder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

USB-A port . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Accessories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

DYMO™ LabelWriter™ 450 USB Label Printer . . . . . . . . . . . . . . . . .12

PR-1 Pedestal Reconditioning Kit . . . . . . . . . . . . . . . . . . . . . . . . . . .12

PV-1 Performance Verification Solution . . . . . . . . . . . . . . . . . . . . . . . 12

Instrument Detection Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

Chapter 2 Instrument Set up. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Update Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Setting Up User Account Control (Optional). . . . . . . . . . . . . . . . . . . . . .16

User Account Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

Security Administration Policies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

For U.S./Canada Support, please contact: . . . . . . . . . . . . . . . . . . . .19

For International Support, please contact: . . . . . . . . . . . . . . . . . . . . .19

Chapter 3 Application Measurement Ranges . . . . . . . . . . . . . . . . . . . . . . . . . .21

Detection Limits for All Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Detection limits for standard applications . . . . . . . . . . . . . . . . . . . . . .21

Detection limits for pre-defined dyes . . . . . . . . . . . . . . . . . . . . . . . . .23

Chapter 4 Nucleic Acid Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

Measure dsDNA, ssDNA or RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

Measure dsDNA, ssDNA or RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

Best practices for nucleic acid measurements . . . . . . . . . . . . . . . . . .27

Nucleic Acid Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

Settings for Nucleic Acid Measurements . . . . . . . . . . . . . . . . . . . . . . 32

Calculations for Nucleic Acid Measurements . . . . . . . . . . . . . . . . . . .32

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Contents

Measure Microarray. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Measure Microarray Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Microarray Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Settings for Microarray Measurements. . . . . . . . . . . . . . . . . . . . . . . .41

Calculations for Microarray Measurements. . . . . . . . . . . . . . . . . . . . . 45

Measure using a Custom Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49

Measure Nucleic Acid using a Custom Factor . . . . . . . . . . . . . . . . . . 49

Custom Factor Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . .51

Settings for Nucleic Acid Measurements using a Custom Factor . . . .52

Detection Limits for Nucleic Acid Measurements using a Custom

Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53

Measure Oligo DNA or Oligo RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55

Measure Oligo DNA or Oligo RNA . . . . . . . . . . . . . . . . . . . . . . . . . . .55

Oligo Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Settings for Oligo DNA and Oligo RNA Measurements. . . . . . . . . . . .60

Detection Limits for Oligo DNA and Oligo RNA Measurements. . . . . . 62

Calculations for Oligo DNA and Oligo RNA Measurements. . . . . . . . . 63

Chapter 5 Protein Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Measure Protein A280. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67

Measure Protein Concentration at A280 . . . . . . . . . . . . . . . . . . . . . .67

Best practices for protein measurements. . . . . . . . . . . . . . . . . . . . . .69

Protein A280 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71

Settings for Protein A280 Measurements . . . . . . . . . . . . . . . . . . . . . . 72

Protein editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75

Detection Limits for Protein A280 Measurements. . . . . . . . . . . . . . . .78

Calculations for Protein A280 Measurements. . . . . . . . . . . . . . . . . . .79

Measure Protein A205. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84

Measure Protein Concentration at A205 . . . . . . . . . . . . . . . . . . . . . .84

Protein A205 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86

Settings for Protein A205 Measurements . . . . . . . . . . . . . . . . . . . . . . 89

Calculations for Protein A205 Measurements. . . . . . . . . . . . . . . . . . .91

Measure Proteins and Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .93

Measure Labeled Protein Samples. . . . . . . . . . . . . . . . . . . . . . . . . . .93

Proteins & Labels Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Settings for Proteins and Labels Measurements. . . . . . . . . . . . . . . . .98

Detection Limits for Proteins and Labels Measurements . . . . . . . . .100

Calculations for Proteins and Labels Measurements . . . . . . . . . . . .100

Measure Protein BCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . .103

Protein BCA Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . .111

Settings for Protein BCA Measurements . . . . . . . . . . . . . . . . . . . . .115

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Contents

Measure Protein Bradford . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . .117

Protein Bradford Reported Results . . . . . . . . . . . . . . . . . . . . . . . . .121

Settings for Protein Bradford Measurements . . . . . . . . . . . . . . . . . . 124

Measure Protein Lowry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . .127

To measure Protein Lowry standards and samples . . . . . . . . . . . . .128

Protein Lowry Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Settings for Protein Lowry Measurements . . . . . . . . . . . . . . . . . . . .132

Measure Protein Pierce 660 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .135

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . .135

To measure Protein Pierce 660 standards and samples. . . . . . . . . . 136

Protein Pierce 660 Reported Results . . . . . . . . . . . . . . . . . . . . . . . .138

Settings for Protein Pierce 660 Measurements. . . . . . . . . . . . . . . . . 141

Chapter 6 Measure OD600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Measure OD600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143

To measure OD600 samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .145

OD600 Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147

Settings for OD600 Measurements . . . . . . . . . . . . . . . . . . . . . . . . .148

Calculations for OD600 Measurements . . . . . . . . . . . . . . . . . . . . . .151

Chapter 7 Custom Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Measure UV-Vis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154

Measure UV-Vis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Best practices for UV-Vis measurements . . . . . . . . . . . . . . . . . . . . .155

UV-Vis Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .156

Settings for UV-Vis Measurements. . . . . . . . . . . . . . . . . . . . . . . . . .159

Measure Custom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161

Measure using a Custom Method . . . . . . . . . . . . . . . . . . . . . . . . . .161

Delete Custom Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .164

Custom Method Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . .165

Manage Custom Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .167

Chapter 8 Measure Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177

Measure Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177

Create Kinetics Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .181

Edit Kinetics Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182

Kinetics Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183

Settings for Kinetic Measurements. . . . . . . . . . . . . . . . . . . . . . . . . .188

Chapter 9 Learning Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .191

Micro-Volume Sampling—How it Works . . . . . . . . . . . . . . . . . . . . . . .192

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Contents

Set Up the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .194

Connect Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .194

Connect an Accessory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .194

Set Up Bluetooth Connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

Set Up Ethernet Connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .199

Set up Wireless Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Assess Instrument Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . .202

Operating Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

Measure a Micro-Volume Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . .204

Best practices for micro-volume measurements. . . . . . . . . . . . . . . .205

Recommended sample volumes . . . . . . . . . . . . . . . . . . . . . . . . . . .206

Measure a Sample Using a Cuvette. . . . . . . . . . . . . . . . . . . . . . . . . . .209

Best practices for cuvette measurements . . . . . . . . . . . . . . . . . . . .210

Prepare Samples and Blanks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .212

Preparing Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .212

Run a Blanking Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .215

Basic Instrument Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .217

NanoDrop One Home Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . .217

NanoDrop One Measurement Screens . . . . . . . . . . . . . . . . . . . . . .220

NanoDrop One General Operations . . . . . . . . . . . . . . . . . . . . . . . . .235

Acclaro Sample Intelligence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .245

Activate Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .245

View Acclaro Sample Intelligence Information. . . . . . . . . . . . . . . . . . 246

Contaminant Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .247

On-Demand Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . .251

Invalid-Results Alerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .252

Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .253

System Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

Network Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .255

Export Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .256

General Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Data Deletion Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .259

PC Control Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .261

PC Control Home Screen overview . . . . . . . . . . . . . . . . . . . . . . . . .261

Control options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .262

Instrument Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .265

Measurement Screen Display Options . . . . . . . . . . . . . . . . . . . . . . .266

Chapter 10 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .267

Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .268

Daily Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

Periodic Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .268

Every 6 Months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .268

Cleaning the Touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

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Contents

Maintaining the Pedestals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .269

Cleaning the Pedestals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .269

Reconditioning the Pedestals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .272

Decontaminating the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . .274

Maintaining the Cuvette Sampling System. . . . . . . . . . . . . . . . . . . . . .276

Instrument Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

Intensity Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .277

Performance Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .279

Pedestal Image Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

Chapter 11 Safety and Operating Precautions . . . . . . . . . . . . . . . . . . . . . . . . .285

Operating Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .286

Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .287

Safety and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .287

When the System Arrives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

Lifting or Moving the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

Electrical Requirements and Safety . . . . . . . . . . . . . . . . . . . . . . . . . 290

Power Cords . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .290

Fire Safety and Burn Hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . .291

Optical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .292

Hazardous Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .292

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Contents

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Page 9

About the Spectrophotometer

NanoDrop OneC Spectrophotometer

Arm

Pedestal

Cuvette holder

Note Locate the instrument away from air vents and exhaust fans to minimize

evaporation

The Thermo Scientific™ NanoDrop™ OneC is a compact, stand-alone UV-Visible spectrophotometer developed for micro-volume analysis of a wide variety of analytes. The patented sample retention system enables the measurement of highly concentrated samples without the need for dilutions.

The NanoDrop One system comes with preloaded software and a touchscreen display. NanoDrop One PC Control software can be installed on a local PC and used to control the instrument and view data. The instrument can be connected to an optional printer with a USB cable or to a remote printer through an Ethernet connection or wireless network.

Note Before operating a NanoDrop One instrument, please read the safety and

operating precautions and then follow their recommendations when using the

instrument.

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1 About the Spectrophotometer

Features

Touchscreen

TheNanoDrop OneC spectrophotometer features the patented micro-volume sample

retention system. The NanoDrop One

dilute samples using standard UV-Visible cuvettes.

TheNanoDrop OneC comes with a built-in, 7-inch high-resolution touchscreen preloaded with easy-to-use instrument control software. The touchscreen can slide left or right to accommodate personal preference, and tilt forward or back for optimal viewing

also features a cuvette holder for analyzing

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Cuvette Holder

Instrument light path

About the Spectrophotometer

Cuvette holder

Features

USB-A port

The NanoDrop OneC includes a cuvette holder for measuring dilute samples, colorimetric assays, cell cultures and kinetic studies. The cuvette system has these features:

• extended lower detection limits

• 37 °C heater option for temperature-sensitive samples and analyses

• micro-stirring option to ensure sample homogeneity and support kinetic studies

For details, see Measure a Sample using a Cuvette.

One USB-A port is located on the front of the instrument and two more USB-A ports are located on instrument back panel.

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1 About the Spectrophotometer

Accessories

This section lists the accessories included for use with the NanoDrop OneC.

DYMO™ LabelWriter™ 450 USB Label Printer

Prints two 5/16-in x 4-in self-adhesive labels for transferring sample data directly into laboratory notebooks or posting on bulletin boards. The software allows printing of

data from each sample measurement or from a group of samples logged and

measured together.

The printer connects to the instrument (front or back) via a USB cable (included).

PR-1 Pedestal Reconditioning Kit

Specially formulated conditioning compound that can be applied to the pedestals to restore them to a hydrophobic state (required to achieve adequate surface tension for accurate sample measurements). The kit includes conditioning compound and applicators. For more information, see Reconditioning the

Pedestals.

PV-1 Performance Verification Solution

Liquid photometric standard used to check instrument performance. For more information, see Performance Verification.

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Page 13

Instrument Detection Limits

About the Spectrophotometer

Instrument Detection Limits

Measurement Location Pathlength (mm)

Upper Detection Limit (10 mm Equivalent Absorbance)

Pedestal 1.0 12.5

0.2 62.5

0.1 150

0.05 300

0.03 550

Cuvette 10 1.5

2 7.5

115

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1 About the Spectrophotometer

This page is intentionally blank.

14 NanoDrop One User Guide Thermo Scientific

Page 15

Instrument Set up

Register Your Instrument

Register your instrument to receive e-mail updates on software and accessories for the NanoDrop One instrument. An Internet connection is required for registration.

To register your instrument

1. Do one of the following:

– From any PC that is connected to the Internet, use any web browser to

navigate to our website.

On the website, locate NanoDrop One Registration and follow the instructions to register the instrument.

Update Software

Quickly and easily download and install the latest NanoDrop One software and release notes from our website. Follow the steps to update or upgrade the software on your local instrument and/or install or update the NanoDrop One software on a personal computer (PC). An Internet connection is required to download software.

To install or update NanoDrop One software on a PC

1. Insert the USB flash drive containing the installer software into an available USB port on your PC, or open the installation folder downloaded from the internet.

2. Launch Start.exe and click Install. The software installer will run.

To install or update NanoDrop One software on the instrument

1. Copy the .zip file with the new software from your computer to a USB storage device. Do not attempt to unzip the folder.

2. Insert the USB device into any USB port on the NanoDrop One instrument.

3. From the instrument Home screen, tap Settings > System > Update Software and choose the latest version of software.

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2 Instrument Set up

Setting Up User Account Control (Optional)

User account control is managed using the Security Administration application. The Thermo Scientific Security Administration software for NanoDrop One may be purchased for instruments used in labs requiring 21 CFR Part 11 compliance. When you launch Security Administration, you will need to enter your Windows log-in information.

User Account Control

Launch the Security Administration application and select NanoDrop One from the directory on the left to reveal Access Control and System Policies.

Access control

Access control is used to grant or deny individual users or groups access to protected features in the instrument application. Add and remove users and groups to the access list and set access rights using the drop-down for each entity.

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System policies

System Policies is used to set options that define the behavior of the client application. See “Security Administration Policies.”

Security Administration Policies

System policies allow you to assign data and method creation and deletion and editing privileges for users and groups.

Launch the Security Administration application and select NanoDrop One->

System Policies

Setting Up User Account Control (Optional)

Instrument Set up

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2 Instrument Set up

Setting Up User Account Control (Optional)

You can add, delete, or edit policy groups and enable or disable the group’s users permission to delete data. Select NanoDrop One-> Access Control.

When you are finished, select Save. Changes will take effect the next time NanoDrop One is launched.

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Technical Support

For U.S./Canada Support, please contact:

Thermo Fisher Scientific 3411 Silverside Road Tatnall Building, Suite 100 Wilmington, DE 19810 U.S.A.

Telephone: 302 479 7707 Toll Free: 1 877 724 7690 (U.S. & Canada only) Fax: 302 792 7155 E-mail: nanodrop@thermofisher.com Website: www.thermofisher.com/nanodrop

Instrument Set up

Technical Support

For International Support, please contact:

Contact your local distributor. For contact information go to:

http://www.thermofisher.com/NanoDropDistributors

If you are experiencing an issue with your system, refer to the troubleshooting information. If the issue persists, contact us. If you are outside the U.S.A. and Canada, please contact your local distributor.

If your instrument requires maintenance or repair, contact us or your local distributor.

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Instrument Set up

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Application Measurement Ranges

Detection Limits for All Applications

Note Detection limits provided in the tables below are approximate and apply to

micro-volume measurements only; they are based on the instrument’s photometric absorbance range (10 mm equivalent) of 0–550 A. For measurements with 10 mm pathlength cuvettes, the photometric absorbance range is 0–1.5 A.

Detection limits for standard applications

Sample Type

dsDNA 2.0 ng/µL (pedestal)

ssDNA 1.3 ng/µL (pedestal)

Lower Detection Limit

0.20 ng/µL (cuvette)

0.13 ng/µL (cuvette)

Upper Detection Limit

27,500 ng/µL (pedestal)

75 ng/µL (cuvette)

18,150 ng/µL (pedestal)

49.5 ng/µL (cuvette)

Typical Reproducibility

±2.0 ng/µL for sample concentrations between 2.0 and 100 ng/µL samples; ±2% for samples >100 ng/µL

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3 Application Measurement Ranges

Detection Limits for All Applications

Sample Type

Lower Detection Limit

RNA 1.6 ng/µL (pedestal)

0.16 ng/µL (cuvette)

DNA Microarray

1.3 ng/µL (pedestal)

(ssDNA)

0.13 ng/µL (cuvette)

Purified BSA by Protein A280

IgG by Protein A280

Purified BSA by

0.06 mg/mL (pedestal)

0.006 mg/mL (cuvette)

0.03 mg/mL (pedestal)

0.003 mg/mL (cuvette)

0.06 mg/mL (pedestal) Proteins & Labels

0.006 mg/mL (cuvette)

Protein BCA 0.2 mg/mL (20:1

reagent/sample volume)

Upper Detection Limit

22,000 ng/µL (pedestal)

Typical Reproducibility

±2.0 ng/µL for sample concentrations between 2.0 and

100 ng/µL samples;

60 ng/µL (cuvette)

495 ng/µL (pedestal)

±2% for samples >100 ng/µL

±2.0 ng/µL for sample concentrations between 2.0 and

49.5 ng/µL (cuvette)

100 ng/µL samples; ±2% for samples >100 ng/µL

825 mg/mL (pedestal)

±0.10 mg/mL (for 0.10–10 mg/mL samples); ±2% for samples >10 mg/mL

402 mg/mL (pedestal)

19 mg/mL (pedestal) ±0.10 mg/mL for 0.10–10 mg/mL

samples

8.0 mg/mL (pedestal)

0.20 mg/mL (cuvette)

2% over entire range

0.01 mg/mL over entire range

0.01 mg/mL (1:1

reagent/sample volume)

Protein Lowry 0.2 mg/mL (pedestal) 4.0 mg/mL (pedestal) 2% over entire range

Protein Bradford 100 µg/mL (50:1

reagent/sample volume)

15 µg/mL (1:1 reagent/sample

8000 µg/mL

100 µg/µL

±25 µg/mL for 100–500 µg/mL samples ±5% for 500–8000 µg/mL samples

±4 µg/mL for 15–50 µg/mL samples ±5% for 50–125 µg/mL samples

volume)

Protein Pierce 660

50 µg/mL (15:1 reagent/sample

2000 µg/mL

±3 µg/mL for 50–125 µg/mL samples ±2% for samples > 125 µg/mL

volume)

±3 µg/mL for 25–125 µg/mL samples

25 µg/mL (7.5:1

1000 µg/mL

±2% for samples >125 µg/mL reagent/sample volume)

Based on five replicates (SD=ng/µL; CV=%)

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Note To minimize instrument error with highly concentrated samples, make

dilutions to ensure that measurements are made within these absorbance limits:

• For micro-volume measurements, maximum absorbance at 260 nm (for nucleic acids) or 280 nm (for proteins) should be less than 62.5 A.

• For measurements with 10 mm pathlength cuvettes, maximum absorbance at 260 nm (or 280 nm for proteins) should be less than 1.5 A, which is approximately 75 ng/µL dsDNA.

Detection limits for pre-defined dyes

Application Measurement Ranges

Detection Limits for All Applications

Sample Type

Cy3, Cy3.5, Alexa Fluor 555, Alexa Fluor

Lower Detection Limit

0.2 pmol/µL

(pedestal)

660

Cy5, Cy5.5, Alexa Fluor 647

Alexa Fluor 488, Alexa Fluor 594

0.12 pmol/µL

(pedestal)

0.4 pmol/µL

(pedestal)

Alexa Fluor 546 0.3 pmol/µL

(pedestal)

Values are approximate

Based on five replicates (SD=ng/µL; CV=%)

Upper Detection

Limit

100 pmol/µL (pedestal)

60 pmol/µL (pedestal)

215 pmol/µL (pedestal)

145 pmol/µL (pedestal)

Typical Reproducibility

±0.20 pmol/µL for sample concentrations between 0.20 and

4.0 pmol/µL; ±2% for samples >4.0 pmol/µL

±0.12 pmol/µL for sample concentrations between 0.12 and

2.4 pmol/µL; ±2% for samples >2.4 pmol/µL

±0.40 pmol/µL for sample concentrations between 0.40 and

8.0 pmol/µL; ±2% for samples >8.0 pmol/µL

±0.30 pmol/µL for sample concentrations between 0.30 and

6.0 pmol/µL; ±2% for samples >6.0 pmol/µL

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Application Measurement Ranges

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Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

Measures the concentration of purified dsDNA, ssDNA or RNA samples that absorb at 260 nm.

Measure dsDNA, ssDNA or RNA

Reported Results

Settings

Detection Limits

Calculations

Measure dsDNA, ssDNA or RNA

Use the dsDNA, ssDNA and RNA applications to quantify purified double-stranded (ds) or single-stranded (ss) DNA or RNA samples. These applications report nucleic acid concentration and two absorbance ratios (A260/A280 and A260/A230). A single-point baseline correction can also be used.

To measure dsDNA, ssDNA or RNA samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

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4 Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

Before you begin...

Before taking pedestal measurements with the NanoDrop One instrument, lift the instrument arm and clean the upper and lower pedestals. At a minimum, wipe the pedestals with a new laboratory wipe. For more information, see Cleaning the

Pedestals.

To measure nucleic acid

1. From the Home screen, select the Nucleic Acids tab and select dsDNA,

2. Specify a baseline correction if desired.

3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm, or

ssDNA or RNA, depending on the samples to be measured.

insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 1-2 µL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

– Pedestal: If Auto-Measure is On, lower arm; if Auto-Measure is off, lower arm

and tap Measure.

– Cuvette: Tap Measure.

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

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Typical nucleic acid spectrum

Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

Comparison of nucleic acid spectra with and without two common contaminants

Best practices for nucleic acid measurements

• Isolate and purify nucleic acid samples before measurement to remove impurities. Depending on the sample, impurities could include DNA, RNA, free nucleotides, proteins, some buffer components and dyes. See Preparing

Samples for more information.

Note Extraction reagents such as guanidine, phenol, and EDTA contribute absorbance between 230 nm and 280 nm and will affect measurement results if present in samples (even residual amounts).

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4 Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

• Ensure the sample absorbance is within the instrument’s absorbance detection

• Blank with the same buffer solution used to resuspend the analyte of interest.

• Run a blanking cycle to assess the absorbance contribution of your buffer

• For micro-volume measurements:

limits.

The blanking solution should be a similar pH and ionic strength as the analyte solution.

solution. If the buffer exhibits strong absorbance at or near the analysis wavelength (typically 260 nm), you may need to choose a different buffer or application. See Choosing and Measuring a Blank for more information.

– Ensure pedestal surfaces are properly cleaned and conditioned.

– If possible, heat highly concentrated or large molecule samples, such as

genomic or lambda DNA, to 63 °C (145 °F) and gently (but thoroughly) vortex before taking a measurement. Avoid introducing bubbles when mixing and pipetting.

– Follow best practices for micro-volume measurements.

– Use a 1-2 µL sample volume. See Recommended Sample Volumes for more

information.

• For cuvette measurements (NanoDrop One

instruments only), use compatible

cuvettes and follow best practices for cuvette measurements.

Related Topics

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Best Practices for Micro-Volume Measurements

• Best Practices for Cuvette Measurements

• Prepare Samples and Blanks

• Basic Instrument Operations

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Nucleic Acid Reported Results

dsDNA measurement screen

For each measured sample, the dsDNA, ssDNA and RNA applications show the UV absorbance spectrum and a summary of the results. Below is an example of the measurement screen of the PC control software:

Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

Application

Sampling method

Sample name of next measurement; select to edit

Measure sample

Run Blank

UV spectrum

End experiment

Menu of options; click to open

Right click graph area to view

options

Mammalian

display

Contaminant Detection

mouse icon to toggle on and off

Menu of table options; click to choose which columns to report

; select

Sample name; select to edit

Nucleic acid concentration

Purity ratios

Click row to select sample and update spectrum.

Measurement screen of PC Control software

Note Micro-volume absorbance measurements and measurements taken with

nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

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4 Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

The following is an example of the measurement screen of the local control:

Menu of options; tap to open

Pinch and zoom to adjust axes; double-tap to reset

Sample name; tap to edit

Nucleic acid concentration

UV spectrum

Swipe screen left to view table with more measurement results

Tap to select unit

Tap to end experiment and export data

Purity ratios

Tap r ow to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Drag tab down/up to see more/less sample data

Measurement screen of NanoDrop One local control software

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dsDNA, ssDNA and RNA reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

Application

Sampling method

Sample name;

tap to edit

Factor Baseline Correction

(shown here if applied)

Date/time measured

nucleic acid concentration

A260/A280

A260/A230 purity ratio

A260 absorbance

A280 absorbance

• sample details (application and sampling method used, that is, pedestal or cuvette)

• sample name

• created on (date sample measurement was taken)

• nucleic acid concentration

• A260/A280

• A260/A230

• A260

• A280

• factor

• baseline correction

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4 Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

Settings for Nucleic Acid Measurements

To show the dsDNA, ssDNA or RNA settings, from the dsDNA, ssDNA or RNA measurement screen, tap > Nucleic Acid Setup.

Setting Available Options Description

Baseline Correction On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

Optional user-defined baseline correction. Can be used to correct for any offset caused by light scattering particulates by subtracting measured absorbance at specified baseline correction wavelength from absorbance values at all wavelengths in sample spectrum. As a result, absorbance of sample spectrum is zero at specified baseline correction wavelength.

Calculations for Nucleic Acid Measurements

The Nucleic Acid applications use a modification of the Beer-Lambert equation (shown at right) to calculate sample concentration where the extinction coefficient and pathlength are combined and referred to as a “factor.”

Extinction Coefficients vs Factors

Using the terms in the Beer-Lambert equation, factor (f) is defined as:

factor (f) = 1/( * b)

where:

 = wavelength-dependent molar extinction coefficient in

ng-cm/µL b = sample pathlength in cm

As a result, analyte concentration (c) is calculated as:

c = A * [1/( * b)]

c = A * f

where: c = analyte concentration in ng/µL A = absorbance in absorbance units (A) f = factor in ng-cm/µL (see below)

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Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

For the dsDNA, ssDNA and RNA applications, the generally accepted factors for nucleic acids are used in conjunction with Beer’s Law to calculate sample concentration. For the Custom Factor application, the user-specified factor is used.

Calculated nucleic acid concentrations are based on the absorbance value at 260 nm, the factor used and the sample pathlength. A single-point baseline correction (or analysis correction) may also be applied.

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

Absorbance values at 260 nm, 280 nm and sometimes 230 nm are used to calculate purity ratios for the measured nucleic acid samples. Purity ratios are sensitive to the presence of contaminants in the sample, such as residual solvents and reagents typically used during sample purification.

Factors Used

• dsDNA (factor = 50 ng-cm/µL)

• ssDNA (factor = 33 ng-cm/µL)

• RNA (factor = 40 ng-cm/µL)

• Custom Factor (user entered factor between

15 ng-cm/µL and 150 ng-cm/µL

Measured Values

Note: For micro-volume absorbance measurements and

measurements taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

A260 absorbance

• Nucleic acid absorbance values are measured at 260 nm using the normalized spectrum. This is the reported A260 value if Baseline Correction is not selected.

• If Baseline Correction is selected, the absorbance value at the correction wavelength is subtracted from the absorbance at 260 nm. The corrected absorbance at 260 nm is reported and used to calculate nucleic acid concentration.

A230 and A280 absorbance

• Normalized and baseline-corrected (if selected) absorbance values at 230 nm and 280 nm are used to calculate A260/A230 and A260/A280 ratios.

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see

General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

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Reported Values

• Nucleic acid concentration. Reported in selected unit (i.e., ng/µL, µg/uL or µg/mL). Calculations are based on modified Beer’s Law equation using corrected nucleic acid absorbance value.

• A260/A280 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 280 nm. An A260/A280 purity ratio of ~1.8 is generally accepted as “pure” for DNA (~2.0 for RNA). Acidic solutions may under represent the reported value by

0.2-0.3; the opposite is true for basic solutions.

• A260/A230 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 230 nm. An A260/A230 purity ratio between 1.8 and 2.2 is generally accepted as “pure” for DNA and RNA.

Note: Although purity ratios are important indicators of sample quality, the best quality indicator quality is functionality in the downstream application of interest (e.g., real-time PCR).

• Factor. Used in conjunction with Beer’s Law to calculate sample concentration

• Contaminant - If a contaminant was identified by the Acclaro software, the contaminant will be displayed in this column.

• A260 absorbance.

• A280 absorbance.

• Baseline correction.

Page 35

Measure Microarray

Measures the concentration of purified nucleic acids that have been labeled with up to two fluorescent dyes for use in downstream microarray applications.

Measure Microarray Samples

Reported Results

Settings

Detection Limits

Calculations

Measure Microarray Samples

Nucleic Acid Applications

Measure Microarray

Use the Microarray application to quantify nucleic acids that have been labeled with up to two fluorescent dyes. The application reports nucleic acid concentration, an A260/A280 ratio and the concentrations and measured absorbance values of the dye(s), allowing detection of dye concentrations as low as 0.2 picomole per microliter.

To measure microarray samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Before you begin...

Pedestals.

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4 Nucleic Acid Applications

Measure Microarray

To measure a microarray sample

1. From the Home screen, select the Nucleic Acids tab and select Microarray.

2. Specify the sample type and factor and the type of dye(s) used.

3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm, or

4. Select Blank and wait for the measurement to complete.

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the

Tip: Select a dye from the pre-defined list or add a custom dye using the

Dye/Chromophore Editor.

insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette light path with the instrument light path.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

blanking cuvette.

6. Pipette 1-2 µL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

– Pedestal: If Auto-Measure is On, lower arm; if Auto-Measure is off, lower arm

and select Measure.

– Cuvette: Select Measure.

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, select End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

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A260 absorbance peak used to calculate nucleic acid concentration

Nucleic Acid Applications

Measure Microarray

Dye absorbance peak used to calculate dye concentration

Typical microarray spectrum

Related Topics

• Best Practices for Nucleic Acid Measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Best Practices for Micro-Volume Measurements

• Best Practices for Cuvette Measurements

• Prepare Samples and Blanks

• Basic Instrument Operations

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4 Nucleic Acid Applications

Measure Microarray

Microarray Reported Results

Microarray measurement screen (Local Control)

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

Menu of options; tap to open

UV-visible spectrum

Pinch and zoom to adjust axes; double-tap to reset

Sample name; tap to edit

Swipe screen left to view table with more measurement results

Nucleic acid concentration

Tap to select unit

Tap to end experiment and export data

Dye concentration(s)

Tap r ow to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Drag tab down/up to see more/less sample data

Note

• A baseline correction is performed at 850 nm (absorbance value at 850 nm is subtracted from absorbance values at all wavelengths in sample spectrum).

• Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

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Microarray reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

• sample details (application used and pedestal or cuvette)

• sample name

• created on (date sample measurement was taken)

• nucleic acid concentration

• A260

• A260/A280

• dye 1/dye 2 concentration

• sample type

Nucleic Acid Applications

Measure Microarray

• analysis correction

• factor

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4 Nucleic Acid Applications

Measure Microarray

Microarray measurement screen (PC Control)

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

Click to select cuvette or pedestal measurement

Nucleic acid concentration

Sample name; Click to edit

UV-Vis Spectrum

Microarray Setup

Run Blank

Measure sample

Dye concentration(s)

Click to end experiment and export data

Menu of options; click to open

Click and Drag to adjust axes; double-click to reset

Click to select unit

Click to select data columns

Click row to select sample and update spectrum; click more rows to overlay spectra

Note

• A baseline correction is performed at 850 nm (absorbance value at 850 nm is subtracted from absorbance values at all wavelengths in sample spectrum).

• Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

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Settings for Microarray Measurements

Microarray settings

The Microarray Setup screen appears after you select the Microarray application from the Nucleic Acids tab on the Home screen. To show the Microarry settings from the Microarray measurement screen, tap > Microarray Setup.

Setting Available Options Description

Nucleic Acid Applications

Measure Microarray

Sample type and Factor

dsDNA (with non-editable factor of 50 ng-cm/µL)

ssDNA (with non-editable factor of 33 ng-cm/µL)

RNA (with non-editable factor of 40 ng-cm/µL)

Oligo DNA with non-editable calculated factor in ng-cm/µL

Oligo RNA with non-editable calculated factor in ng-cm/µL

Custom (with user-specified factor in ng-cm/µL)

Widely accepted value for double-stranded DNA

Widely accepted value for single-stranded DNA

Widely accepted value for RNA

Factor calculated from user-defined DNA base sequence. When selected, available DNA base units (i.e., G, A, T, C) appear as keys. Define sequence by tapping appropriate keys. Factor is calculated automatically based on widely accepted value for each base unit.

Factor calculated from user-defined RNA base sequence. When selected, available RNA base units (i.e., G, A, U, C) appear as keys. Define sequence by tapping appropriate keys. Factor is calculated automatically based on widely accepted value for each base unit.

Enter factor between 15 ng-cm/µL and 150 ng-cm/µL

Dye 1/Dye 2

Type

Cy3, 5, 3.5, or 5.5, Alexa Fluor 488, 546, 555, 594, 647, or 660

Thermo Scientific NanoDrop One User Guide 41

Select pre-defined dye(s) used to label sample material, or one that has been added using Dye Editor.

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4 Nucleic Acid Applications

Measure Microarray

Setting Available Options Description

Dye 1/Dye 2 Unit picomoles/microliter (pmol/uL),

Select unit for reporting dye concentrations

micromoles (uM), or millimoles (mM)

Analysis Correction

On or off

Enter analysis correction wavelength in nm or use default value (340 nm)

Corrects sample absorbance measurement for any offset caused by light scattering particulates by subtracting absorbance value at specified analysis correction wavelength from absorbance value at analysis wavelength. Corrected value is used to calculate sample concentration.

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Analysis Correction.

To add a custom dye or edit the list of available dyes, use the Dye/Chromophore Editor.

The Analysis Correction affects the calculation for nucleic acid concentration only.

Dye/chromophore editor

Use the Dye/Chromophore Editor to add a custom dye to the list of available dyes in

Microarray Setup or Proteins & Labels Setup. You can also specify which dyes are

available in that list.

To access the Dye/Chromophore Editor:

• from the Home screen, select Settings > Dye Editor

• from the Microarray or Proteins & Labels measurement screen, tap >

Settings > Dye Editor

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Dye Editor

Nucleic Acid Applications

Measure Microarray

Locked dye (pre-defined; cannot be edited or deleted)

Tap to add custom dye

Tap to edit selected custom dye

Tap to delete selected custom dye

Selected dyes (will appear in Dye1 and

Custom dye (user-defined; can be edited or deleted)

Dye2 lists in Microarray Setup or Proteins & Labels Setup)

Tap to close Dye Editor

These operations are available from the Dye/Chromophore Editor:

Add or remove a dye

To add or remove a dye from the Dye1 or Dye2 drop-down list in Microarray Setup or

Proteins & Labels Setup:

– select or deselect corresponding checkbox

Add custom dye

– tap to show New Dye box

– enter unique Name for new dye (tap field to display keyboard, tap Done key

to close keyboard)

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4 Nucleic Acid Applications

Measure Microarray

– select default Unit that will be used to display dye concentration

– enter dye’s Extinction Coefficient (or molar absorptivity constant) in

L/mole-cm (typically provided by dye manufacturer)

– specify Wavelength in nm (between 350 nm and 850 nm) that will be used

to measure dye’s absorbance

– specify dye’s correction values at 260 nm and 280 nm

– tap Add Dye

Note To determine dye correction values (if not available from dye manufacturer):

– use instrument to measure pure dye and note absorbance at 260 nm,

280 nm and at analysis wavelength for dye (see above)

– calculate ratio of A

260/Adye wavelength

and enter that value for 260 nm

Correction

– calculate ratio of A

280/Adye wavelength

and enter that value for 280 nm

Correction

When a custom dye is selected before a measurement, the dye’s absorbance and concentration values are reported and the corrections are applied to the measured sample absorbance values, and to the resulting sample concentrations and purity ratios.

Edit custom dye

Tip Dyes pre-defined in the software cannot be edited.

– tap to select custom dye

– tap

– edit any entries or settings

– tap Save Dye

Delete custom dye

Tip Dyes pre-defined in the software cannot be deleted.

– tap to select custom dye

– tap

NOTICE Deleting a custom dye permanently removes the dye and all associated information from the software.

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Calculations for Microarray Measurements

Nucleic Acid Applications

Measure Microarray

As with the other nucleic acid applications, the Microarray application uses a

modification of the Beer-Lambert equation

to calculate sample concentration where the extinction coefficient and pathlength are combined and referred to as a “factor.” The Microarray application offers six options (shown at right) for selecting an appropriate factor for each measured sample, to be used in conjunction with Beer’s Law to calculate sample concentration.

If the factor is known, choose the Custom Factor option and enter the factor in ng-cm/µL. Otherwise, choose the option that best matches the sample solution.

Tip: Ideally, the factor or extinction coefficient should be determined empirically using a solution of the study nucleic acid at a known concentration using the same buffer.

Available Options for Factors

• dsDNA (factor = 50 ng-cm/µL)

• ssDNA (factor = 33 ng-cm/µL)

• RNA (factor = 40 ng-cm/µL)

• Oligo DNA (calculated from user entered DNA

nucleotide sequence)

• Oligo RNA (calculated from user entered RNA

nucleotide sequence)

• Custom Factor (user entered factor between

15 ng-cm/µL and 150 ng-cm/µL

Note: See Sample Type for more information.

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4 Nucleic Acid Applications

Measure Microarray

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

Measured Values

A260 absorbance

Note: The absorbance value at 850 nm is subtracted from all

wavelengths in the spectrum. As a result, the absorbance at 850 nm is zero in the displayed spectra. Also, for micro-volume absorbance measurements and measurements taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

• Nucleic acid absorbance values for all Microarray sample

types are measured at 260 nm using the 850-corrected

and normalized spectrum.

• If Analysis Correction is selected, the absorbance value at the correction wavelength is subtracted from the absorbance at 260 nm.

• If one or more dyes are selected, the dye correction

values at 260 nm are also subtracted from the

absorbance at 260 nm.

• The final corrected absorbance at 260 nm is reported and used to calculate sample concentration.

A280 absorbance

• 850-corrected and normalized absorbance value at 280 nm (minus the A280 dye correction) is used to calculate an A260/A280 ratio.

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Nucleic Acid Applications

Measure Microarray

Dye concentrations are calculated from the absorbance value at the dye’s analysis wavelength, the dye’s extinction coefficient, and the sample pathlength. A sloped-line dye correction may also be used.

Dye absorbance

• Dye absorbance values are measured at specific wavelengths. See Dye/Chromophore Editor for analysis wavelengths used.

• If Sloping Dye Correction is selected, a linear baseline is drawn between 400 nm and 850 nm and, for each dye, the absorbance value of the sloping baseline is subtracted from the absorbance value at each dye’s analysis wavelength. Baseline-corrected dye absorbance values are reported and used to calculate dye concentrations.

Dye correction

• Pre-defined dyes have known correction values for A260 and A280. See Dye/Chromophore Editor for correction values used.

• A260 dye corrections are subtracted from the A260

absorbance value used to calculate nucleic acid

concentration, and from the A260 absorbance value used to calculate the A260/A280 purity ratio.

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see

General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

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4 Nucleic Acid Applications

Measure Microarray

Reported Values

• Nucleic acid concentration. Reported in selected unit (i.e., ng/µL, µg/uL or µg/mL). Calculations are based on modified Beer’s Law equation using corrected nucleic acid absorbance value.

0.2-0.3; the opposite is true for basic solutions.

• Dye1/Dye2 concentration. Reported in pmol/µL. Calculations are based on Beer’s Law equation using (sloping) baseline-corrected dye absorbance value(s).

Note: Although purity ratios are important indicators of sample quality, the best indicator of DNA or RNA quality is functionality in the downstream application of interest (e.g., microarray).

Related Topics

• Calculations for Nucleic Acid Measurements

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Measure using a Custom Factor

Measures the concentration of purified nucleic acids using a custom factor for the calculations.

Measure using Custom Factor

Reported Results

Settings

Detection Limits

Calculations

Measure Nucleic Acid using a Custom Factor

Nucleic Acid Applications

Measure using a Custom Factor

Use the Custom Factor application to quantify purified DNA or RNA samples that absorb at 260 nm with a user-defined extinction coefficient or factor. The application reports nucleic acid concentration and two absorbance ratios (A260/A280 and A260/A230). A single-point baseline correction can also be used.

To measure nucleic acid samples using a custom factor

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Before you begin...

Pedestals.

To measure using a custom factor

1. From the Home screen, select the Nucleic Acids tab and select Custom Factor.

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4 Nucleic Acid Applications

Measure using a Custom Factor

2. Enter the factor to be used for the calculations and specify a baseline correction

3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm, or

4. Select Blank and wait for the measurement to complete.

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the

6. Pipette 1-2 µL sample solution onto the pedestal and lower the arm, or insert the

7. Start the sample measurement:

if desired.

insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette light path with the instrument light path.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

blanking cuvette.

sample cuvette into the cuvette holder.

– Pedestal: If Auto-Measure is On, lower arm; if Auto-Measure is off, lower arm

and tap Measure.

– Cuvette: Tap Measure.

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

Typical nucleic acid spectrum

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Related Topics

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Best Practices for Micro-Volume Measurements

• Best Practices for Cuvette Measurements

• Prepare Samples and Blanks

• Basic Instrument Operations

Custom Factor Reported Results

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

Nucleic Acid Applications

Measure using a Custom Factor

Note The Custom Factor measurement screen is identical to the measurement

screen for the other nucleic acid applications except the Custom Factor is

reported in the lower left corner (see image below).

Custom factor used to calculate nucleic acid concentration

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4 Nucleic Acid Applications

Measure using a Custom Factor

Related Topics

• Basic Instrument Operations

• Nucleic Acid Reported Results

• Nucleic Acid Calculations

Settings for Nucleic Acid Measurements using a Custom Factor

To show the Custom Factor settings, from the local control, tap > Custom Factor Setup.

When using the PC Control software, from the Custom Factor measurement screen, select the settings icon to view the Custom Factor Setup.

Setting Available Options Description

Custom Factor Enter an integer value

between 15 ng-cm/µL and 150 ng-cm/µL

Baseline Correction On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

Constant used to calculate nucleic acid concentration in

modified Beer’s Law equation. Based on extinction

coefficient and pathlength:



f = 1/(

where: f= factor

260

* b))

 = molar extinction coefficient at 260 nm in ng-cm/µL

b = sample pathlength in cm (1 cm for nucleic acids measured with the NanoDrop One instruments)

NOTE: Baseline correction is selected from the measurement screen of the PC control software and is not shown in the Custom Factor Setup.

Related Topics

• Instrument Settings

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Nucleic Acid Applications

Measure using a Custom Factor

Detection Limits for Nucleic Acid Measurements using a Custom Factor

The lower detection limits and reproducibility specifications for nucleic acids are provided here. The upper detection limits are dependent on the upper absorbance

limit of the instrument and the user-defined extinction coefficients.

To calculate upper detection limits for nucleic acid samples

To calculate upper detection limits in ng/µL, use the following equation:

(upper absorbance limit

For example, for a sample measurement using an extinction coefficient of 55, the equation looks like this:

(550 AU * 55 ng-cm/µL) = 30,250 ng/µL

Note For measurements with 10 mm pathlength cuvettes, the upper absorbance limit is 1.5 AU, which is approximately 75 ng/µL for dsDNA.

instrumen

* extinction coefficient

sample

)

Related Topics

• Detection Limits for All Applications

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Measure Oligo DNA or Oligo RNA

Measures the concentration of purified ssDNA or RNA oligonucleotides that absorb at 260 nm.

Measure Oligo DNA or RNA

Reported Results

Settings

Detection Limits

Calculations

Measure Oligo DNA or Oligo RNA

Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

Use the Oligo DNA and Oligo RNA applications to quantify oligonucleotides that absorb at 260 nm. Molar extinction coefficients are calculated automatically based on the user-defined base sequence of the sample. These applications report nucleic acid concentration and two absorbance ratios (A260/A280 and A260/A230). A single-point baseline correction can also be used.

Note If the oligonucleotide has been modified, for example with a fluorophore dye, check with the oligo manufacturer to determine if the modification contributes absorbance at 260 nm. If it does, we recommend using the

Microarray application to quantify nucleic acid concentration. The Microarray

application includes a correction to remove any absorbance contribution due to the dye from the oligo quantification result.

To measure Oligo DNA or Oligo RNA samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Before you begin...

Pedestals.

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4 Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

To measure an oligonucleotide sample

1. From the Home screen, select the Nucleic Acids tab and select either Oligo DNA or Oligo RNA, as needed.

2. Specify the Oligo base sequence and a baseline correction if desired.

3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 1-2 µL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

– Pedestal: If Auto-Measure is On, lower arm; if Auto-Measure is off, lower arm

and tap Measure.

– Cuvette: Tap Measure.

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

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Measure Oligo DNA or Oligo RNA

Example Oligo DNA spectrum

Nucleic Acid Applications

Related Topics

• Best Practices for Nucleic Acid Measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Best Practices for Micro-Volume Measurements

• Best Practices for Cuvette Measurements

• Prepare Samples and Blanks

• Basic Instrument Operations

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4 Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

Oligo Reported Results

Oligo DNA measurement screen (Local Control)

For each measured sample, the Oligo DNA and Oligo RNA applications show the UV absorbance spectrum and a summary of the results. Here is an example:

Menu of options; tap to open

tap to edit

Nucleic acid concentration

UV spectrum

Tap to select unitSample name;

Purity ratios

Tap row to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample

row to view measure ment details.

Pinch and zoom to adjust axes; double-tap to reset

Measured oligo: TTT TTT TTT TTT TTT TTT TTT TTT

Swipe screen left to view table with more measurement results

Tap to end experiment and export data

Drag tab down/up to see more/less sample data

Note Micro-volume absorbance measurements and measurements taken with

nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

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Oligo DNA and Oligo RNA reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

• sample details (application and sampling method used, i.e., pedestal or cuvette)

• sample name

• created on (date sample measurement was taken)

• nucleic acid concentration

• A260/A280

• A260/A230

• A260

• A280

Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

• factor

• oligo sequence

• baseline correction

• stirrer status

Note The five nucleotides that comprise DNA and RNA exhibit widely varying A260/A280 ratios. See Oligo Purity Ratios for more information.

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4 Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

Oligo DNA and RNA measurement screen (PC Control)

For each measured sample, the Oligo DNA and Oligo RNA applications show the UV absorbance spectrum reported results. Here is an example:

Click to select cuvette or pedestal measurement

Sample name; Click to edit

Oligo RNA/RNA Setup

Run Blank

Measure sample

Click to end experiment and export data

Menu of options; click to open

Nucleic acid concentration

UV spectrum

Purity ratios

Related Topics

• Basic Instrument Operations

• Oligo Calculations

Click and Drag to adjust axes; double-click to reset

Click to select unit

Click row to select sample and update spectrum; click more rows to overlay spectra

Settings for Oligo DNA and Oligo RNA Measurements

The Oligo setup screen appears after you select the Oligo DNA or Oligo RNA application from the Nucleic Acids tab on the Home screen.

In local instrument control, from the Oligo measurement screen, tap >

Oligo DNA Setup (or Oligo RNA Setup).

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From the PC control software, from the Oligo DNA or RNA measurement screen, select the settings icon to view Oligo DNA Setup or Oligo RNA Setup.

Setting Available Options Description

Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

Oligo Base Sequence

for DNA: Use the G, A, T and C keys to specify the DNA base sequence

for RNA: Use the G, A, U and C keys to specify the RNA base sequence

Specify your DNA or RNA base sequence. Tap or click the corresponding keys:

Add guanine

Add adenine

Add thymine (DNA) or uracil (RNA)

From the PC control software, you can also enter base sequence using the keyboard, or by copy and pasting a sequence from another application.

Each time a base is added to the sequence, the software calculates the following:

• Factor. Constant used to calculate oligonucleotide

concentration in modified Beer’s Law equation. Based on extinction coefficient and pathlength:

Add cytosine

Remove most recent base (seen in local instrument control)



f = 1/(

where: f= factor

260

* b)

 = molar extinction coefficient at 260 nm in ng-cm/µL

b = sample pathlength in cm (0.1 cm for nucleic acids measured with the NanoDrop One instrument)

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4 Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

Setting Available Options Description

• Molecular Weight of oligo calculated from user-defined

base sequence.

• Number of Bases entered.

• Molar Ext. Coefficient (260 nm). Molar extinction

coefficient of oligo (in ng-cm/µL) at 260 nm calculated from entered base sequence.

• %GC. Percentage of guanine and cytosine residues in

total number of bases entered.

Baseline Correction

On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

Corrects for any offset caused by light scattering particulates by subtracting measured absorbance at specified baseline correction wavelength from absorbance values at all wavelengths in sample spectrum. As a result, absorbance of sample spectrum is zero at specified baseline correction wavelength.

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Baseline Correction.

Related Topics

• Instrument Settings

Detection Limits for Oligo DNA and Oligo RNA Measurements

The lower detection limits and reproducibility specifications for the oligonucleotide sample types (ssDNA and RNA) are provided here. The upper detection limits are dependent on the upper absorbance limit of the instrument and the extinction coefficients for the user-defined base sequences.

To calculate upper detection limits for nucleic acid samples

To calculate upper detection limits in ng/µL, use the following equation:

(upper absorbance limit

For example, for a sample measurement using an extinction coefficient of 55, the equation looks like this:

(550 AU * 55 ng-cm/µL) = 30,250 ng/µL

Note For measurements with 10 mm pathlength cuvettes, the upper absorbance limit is 1.5 AU, which is approximately 75 ng/µL for dsDNA.

62 NanoDrop One User Guide Thermo Scientific

instrumen

* extinction coefficient

sample

)

Page 63

Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

Calculations for Oligo DNA and Oligo RNA Measurements

As with the other nucleic acid applications, the Oligo applications use the Beer-Lambert

equation to correlate absorbance with

concentration based on the sample’s extinction coefficient and pathlength. Because oligonucleotides are short, single-stranded molecules (or longer molecules of repeating sequences), their spectrum and extinction

coefficient () are closely dependent on base

composition and sequence.

(The generally accepted extinction coefficients and factors for single-stranded DNA and RNA provide a reasonable estimate for natural, essentially randomized, sequences but not for short, synthetic oligo sequences.) To ensure the most accurate results, we use the exact value of



to calculate oligonucleotide concentration.

260

The NanoDrop software allows you to specify the base sequence of an oligonucleotide before it is measured. For any entered base sequence, the software uses the equation at the right to calculate the extinction coefficient.

Tip: The extinction coefficient is wavelength specific for each oligonucleotide and can be affected by buffer type, ionic strength and pH.

Extinction Coefficients for Oligonucleotides

The software uses the nearest neighbor method and the following formula to calculate molar extinction coefficients for specific oligonucleotide base sequences:





where:



260

N1–



N1–



–



 = molar extinction coefficient in L/mole-cm 

= 

nearest neighbor



= 

individual bases



= 

modifications, such as fluorescent dyes

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4 Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

Measured Values

A260 absorbance

Note: For micro-volume absorbance measurements and

measurements taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

• Nucleic acid absorbance values are measured at 260 nm using the normalized spectrum. This is the reported A260 value if Baseline Correction is not selected.

• If Baseline Correction is selected, the absorbance value at the correction wavelength is subtracted from the sample absorbance at 260 nm. The corrected absorbance at 260 nm is reported and used to calculate nucleic acid concentration.

A230, A280 absorbance

• Normalized absorbance values at 230 nm, 260 nm and 280 nm are used to calculate A260/A230 and A260/A280 ratios.

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and

0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see

General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

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Nucleic Acid Applications

Measure Oligo DNA or Oligo RNA

The five nucleotides that comprise DNA and RNA exhibit widely varying A260/A280 ratios. Estimated A260/A280 ratios for each independently measured nucleotide are provided below:

Guanine: 1.15 Adenine: 4.50 Cytosine: 1.51 Uracil: 4.00 Thymine: 1.47

The A260/A280 ratio for a specific nucleic acid sequence is approximately equal to the weighted average of the A260/A280 ratios for the four nucleotides present.

Note: RNA will typically have a higher 260/280 ratio due to the higher ratio of Uracil compared to that of Thymine.

Reported Values

• Nucleic acid concentration. Reported in selected unit (i.e., ng/µL, µg/uL or µg/mL). Calculations are based on modified Beer’s Law equation using corrected nucleic acid absorbance value.

• A260/A280 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 280 nm.

• A260/A230 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 230 nm.

Note: The traditional purity ratios (A260/A280 and A260/A230), which are used as indicators of the presence of various contaminants in nucleic acid samples, do not apply for oligonucleotides because the shapes of their spectra are highly dependent on their base compositions. See side bar for more information.

Related Topics

• Calculations for Nucleic Acid Measurements

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Protein Applications

Measure Protein A280

Measures the concentration of purified protein samples that absorb at 280 nm.

Measure A280 Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Protein Concentration at A280

Use the Protein A280 application to quantify purified protein samples that contain amino acids such as tryptophan or tyrosine, or cys-cys disulfide bonds, which exhibit absorbance at 280 nm. This application reports protein concentration measured at 280 nm and one absorbance ratio (A260/A280). A single-point baseline correction can also be used. This application does not require a standard curve.

Note If your samples contain mainly peptide bonds and little or no amino acids, use the Protein A205 application instead of Protein A280.

To measure Protein A280 samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

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5 Protein Applications

Measure Protein A280

Before you begin...

Pedestals.

To measure a Protein A280 sample

1. From the Home screen, select the Proteins tab and select Protein A280.

2. Specify a sample type and baseline correction if desired.

3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 2 µL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

– Pedestal: If Auto-Measure is On, lower arm; if Auto-Measure is off, lower arm

and tap Measure.

– Cuvette: Tap Measure

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

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High concentration BSA sample

Protein Applications

Measure Protein A280

Low concentration BSA sample

Best practices for protein measurements

• Isolate and purify protein samples before measurement to remove impurities. Depending on the sample, impurities could include DNA, RNA and some buffer components. See Preparing Samples for more information.

Note Extraction reagents that contribute absorbance between 200 nm and 280 nm will affect measurement results if present in samples (even residual amounts).

• Ensure the sample absorbance is within the instrument’s absorbance detection

limits.

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5 Protein Applications

Measure Protein A280

• Choosing a blank:

– For the Protein A280, Protein A205, and Proteins & Labels applications,

blank with the same buffer solution used to resuspend the analyte of interest. The blanking solution should be a similar pH and ionic strength as the analyte solution.

– For the Protein BCA, Protein Bradford, and Protein Lowry applications, blank

with deionized water (DI H

O).

– For the Protein Pierce 660 application, blank with the reference solution used

to make the standard curve (reference solution should contain none of the standard protein stock). For more information, see Working with standard

curves.

• Run a blanking cycle to assess the absorbance contribution of your buffer solution. If the buffer exhibits strong absorbance at or near the analysis wavelength (typically 280 nm or 205 nm), you may need to choose a different buffer or application, such as a colorimetric assay (for example, BCA or Pierce 660). See Choosing and Measuring a Blank for more information.

Note Buffers such as Triton X, RIPA, and NDSB contribute significant absorbance and are not compatible with direct A280 or A205 measurements.

• For micro-volume measurements:

– Ensure pedestal surfaces are properly cleaned and conditioned. (Proteins

tend to stick to pedestal surfaces.)

– Gently (but thoroughly) vortex samples before taking a measurement. Avoid

introducing bubbles when mixing and pipetting.

– Follow best practices for micro-volume measurements.

– Use a 2 µL sample volume. See Recommended Sample Volumes for more

information.

• For cuvette measurements (NanoDrop One

instruments only), use compatible

cuvettes and follow best practices for cuvette measurements.

Related Topics

• Best practices for protein measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Prepare Samples and Blanks

• Basic Instrument Operations

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Protein A280 Reported Results

Protein A280 measurement screen (Local Control)

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example of the Local control screen:

Protein Applications

Measure Protein A280

tap to open

Sample name; tap to edit

UV spectrum

Protein concentration

Tap to select unitMenu of options;

Absorbance at

280 nm

Purity ratio

Tap r ow to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Pinch and zoom to adjust axes; double-tap to reset

Swipe screen left to view table with more measurement results

Tap to end experiment and export data

Drag tab down/up to see more/less sample data

Note Micro-volume absorbance measurements and measurements taken with

nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

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5 Protein Applications

Measure Protein A280

Protein A280 reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

Application

Baseline correction wavelength

Sampling method

Baseline correction absorbance

Sample name;

tap to edit

Date/time measured

Protein conc.

Absorbance at 280 nm

Purity ratio

Sample type

Related Topics

• Basic Instrument Operations

• Protein A280 Calculations

Settings for Protein A280 Measurements

To show the Protein A280 settings, in local instrument control, from the Protein A280 measurement screen, tap > Protein A280 Setup.

From the PC control software, from the Protein A280 measurement screen, select the settings icon to view Protein A280 Setup.

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Protein A280 settings

The Protein A280 application provides a variety of sample type options for purified protein analysis.

Each sample type applies a unique extinction coefficient to the protein calculations. If the extinction coefficient of the sample is known, choose the (mass) option and enter the value. Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution. If you only need a rough estimate of protein concentration and the sample extinction coefficient is unknown, select the 1 Abs=1 mg/mL sample type option.

Protein Applications

Measure Protein A280

 + MW (molar) or 1%

Tip Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

Setting Available Options

Sample

type

1 Abs = 1 mg/mL General reference Recommended when extinction coefficient is

BSA 6.7 Calculates BSA (Bovine Serum Albumin)

Mass Ext. Coefficient (L/gm-cm)

Description

unknown and rough estimate of protein concentration is acceptable for a solution with no other interfering substances. Assumes

0.1% (1 mg/mL) protein solution produces

1.0A at 280 nm (where pathlength is 10 mm),

1% = 10.

i.e.,

protein concentration using mass extinction coefficient (

) of 6.7 L/gm-cm at 280 nm for

1% (i.e., 10 mg/mL) BSA solution. Assuming MW is 66,400 daltons (Da), molar extinction coefficient at 280 nm for BSA is approximately 43,824 M

-1cm-1

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Measure Protein A280

Setting Available Options

IgG 13.7 Suitable for most mammalian antibodies (i.e.,

Lysozyme 26.4 Calculates lysozyme protein concentration

Mass Ext. Coefficient (L/gm-cm)

Description

immunoglobulin G or IgG). Calculates protein concentration using mass extinction coefficient ( 1% (i.e., 10 mg/mL) IgG solution. Assuming MW is 150,000 Da, molar extinction coefficient at 280 nm for IgG is approximately 210,000 M

using mass extinction coefficient (

26.4 L/gm-cm at 280 nm for 1% (i.e., 10 mg/mL) lysozyme solution. Assumes molar extinction coefficient for egg white lysozyme ranges between 36,000 M

) of 13.7 L/gm-cm at 280 nm for

-1cm-1

) of

-1cm-1

and 39,000 M-1cm-1.

Other protein

 + MW)

(

Other protein

1%)

(

To add or edit a custom protein, use Protein Editor.

User entered molar extinction coefficient and molecular weight

User entered mass extinction coefficient

Assumes protein has known molar extinction coefficient ( where:



(

molar

Enter MW in kiloDaltons (kDa) and molar extinction coefficient ( 1000 (i.e., with molar extinction coefficient of 210,000

-1cm-1

Assumes protein has known mass extinction coefficient (). Enter mass extinction coefficient in L/gm-cm for 10 mg/mL ( protein solution.

) and molecular weight (MW),

)*10=(

percent

)*(MW

) in M

protein

-1cm-1

)

/1000). For example, for protein

, enter 210.

divided by

1%)

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Setting Available Options

Mass Ext. Coefficient (L/gm-cm)

Description

Protein Applications

Measure Protein A280

Baseline

On or off

Correction

Enter baseline correction wavelength in nm or use default value (340 nm)

Protein editor

N/A Corrects for any offset caused by light

scattering particulates by subtracting measured absorbance at specified baseline correction wavelength from absorbance values at all wavelengths in sample spectrum. As a result, absorbance of sample spectrum is zero at specified baseline correction wavelength.

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Baseline Correction.

Use the Protein Editor to add a custom protein to the list of available protein sample types in Protein A280 Setup and Proteins & Labels Setup.

To access the Protein Editor:

• From the PC control software Home screen, select Settings > Protein Editor.

PC Control software Protein Editor

Click to add custom protein

Click to edit selected custom protein

Click to delete selected custom

Custom proteins (will appear in Sample Type list in Protein A280 Setup and Proteins & Labels Setup)

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5 Protein Applications

Measure Protein A280

• From the local control Home screen, tap > Protein Editor. Alternatively, from the Protein A280 or Proteins & Labels measurement screen, tap >

Settings > Protein Editor.

Local Control Protein Editor

Tap to add custom protein

Custom proteins (will appear in Sample Type list in Protein A280 Setup and Proteins & Labels Setup)

Tap to edit selected custom protein

Tap to delete selected custom protein

Tap to close Protein Editor

These operations are available from the Protein Editor:

Add custom protein

1. In Protein Editor, select or to show the New Protein Type

box.

2. Enter a unique Name for the new protein (from local control, tap field to display keyboard, tap Done key to close keyboard).

3. Enter a Description for the new protein.

4. Specify whether to enter Molar Extinction coefficient or Mass Extinction coefficient for custom protein.

– If Mass Extinction coefficient is selected, enter mass extinction

coefficient in L/gm-cm for 10 mg/mL (

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1%) protein solution.

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Protein Applications

Measure Protein A280

Tap a field to show keyboard; to close, tap Done key

– if Molar Extinction is selected,

– enter molar extinction coefficient (

/1000). For example, for protein with molar extinction coefficient

is, of 210,000 M

-1cm-1

, enter 210.

) in M

-1cm-1

divided by 1000 (that

– Enter molecular weight (MW) in kiloDaltons (kDa)

5. From local control, tap OK to close the New Protein Type box. From PC control software, select Save.

6. Enter your password to sign the changes if prompted.

Enter your password to sign the changes

The new custom protein appears in the Type list in Protein A280 Setup and Proteins & Labels Setup.

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5 Protein Applications

Measure Protein A280

Edit custom protein

1. In Protein Editor, tap to select custom protein

2. Tap to show the Edit Protein Type box

3. Edit any entries or settings

4. Tap OK

Delete custom protein

1. In Protein Editor, tap to select a custom protein to delete

2. Tap

Note Deleting a custom protein permanently removes the protein and all associated information from the software.

Detection Limits for Protein A280 Measurements

Detection limits and reproducibility specifications for purified BSA proteins are provided here. The BSA lower detection limit and reproducibility values apply to any protein sample type. The upper detection limits are dependent on the upper

absorbance limit of the instrument and the sample’s extinction coefficient.

To calculate upper detection limits for other (non-BSA) protein sample types

To calculate upper detection limits in ng/µL for proteins, use the following equation:

(upper absorbance limit

For example, if the sample’s mass extinction coefficient at 280 nm is 6.7 for a 1% (10 mg/mL) solution, the equation looks like this:

(550 / 6.7) * 10 = 824.6 (or ~825)

instrumen

/mass extinction coefficient

sample

) * 10

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Calculations for Protein A280 Measurements

Protein Applications

Measure Protein A280

The Protein A280 application uses the

Beer-Lambert equation to correlate

absorbance with concentration. Solving Beer’s law for concentration yields the equation at the right.

The extinction coefficient of a peptide or protein is related to its tryptophan (W), tyrosin (Y) and cysteine (C) amino acid composition.

Tip: The extinction coefficient is wavelength specific for each protein and can be affected by buffer type, ionic strength and pH.

Beer-Lambert Equation (solved for concentration)

c = A / ( * b)

where:

A = UV absorbance in absorbance units (AU)

 = wavelength-dependent molar absorptivity coefficient (or

extinction coefficient) in liter/mol-cm

b = pathlength in cm

c = analyte concentration in moles/liter or molarity (M)

Note: Dividing the measured absorbance of a sample solution by its molar extinction coefficient yields the molar concentration of the sample. See Published Extinction

Coefficients for more information regarding molar vs. mass

concentration values.

Extinction Coefficients for Proteins

At 280 nm, the extinction coefficient is approximated by the weighted sum of the 280 nm molar extinction coefficients of the three constituent amino acids, as described in this equation:

 = (nW * 5500) + (nY * 1490) + (nC * 125)

where:

 = molar extinction coefficient

n = number of each amino acid residue 5500, 1490 and 125 = amino acid molar absorptivities at

280 nm

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5 Protein Applications

Measure Protein A280

This application offers six options (shown at right) for selecting an appropriate extinction coefficient for each measured sample, to be used in conjunction with Beer’s Law to calculate sample concentration.

If the extinction coefficient of the sample is known, choose the

 + MW (molar) or

1% (mass) option and enter the value.

Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution.

Tip: Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

Most sources report extinction coefficients for proteins measured at or near 280 nm in phosphate or other physiologic buffer. These values provide sufficient accuracy for routine assessments of protein concentration.

Available Options for Extinction Coefficient

• 1 Abs = 1 mg/mL, where sample type and/or ext.

coefficient is unknown (produces rough estimate of protein concentration)

• BSA (Bovine Serum Albumin, 6.7 L/gm-cm)

• IgG (any mammalian antibody, 13.7 L/gm-cm)

• Lysozyme (egg white lysozyme, 26.4 L/gm-cm)

• Other protein (

 + MW), user-specified molar ext.

coefficient

• Other protein (

1%), user-specified mass ext.

coefficient

•

Note: See Sample Type for details.

Published Extinction Coefficients

Published extinction coefficients for proteins may be reported as:

• wavelength-dependent molar absorptivity (or extinction) coefficient (

) with units of M

-1cm-1

• percent solution extinction coefficient (1%) with units of (g/100 mL)

-1cm-1

(i.e., 1% or 1 g/100 mL solution

measured in a 1 cm cuvette)

• protein absorbance values for 0.1% (i.e., 1 mg/mL) solutions

Tip: Assess published values carefully to ensure unit of measure is applied correctly.

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Protein Applications

Measure Protein A280

The equation at the right shows the relationship between molar extinction

coefficient ( coefficient (1%).

To determine concentration (c) of a sample in mg/mL, use the equation at the right and a conversion factor of 10.

Tip: The NanoDrop One software includes the conversion factor when reporting protein concentrations.

) and percent extinction

molar

Conversions Between 

(

Example: To determine percent solution extinction coefficient

1%) for a protein that has a molar extinction coefficient of

( 43,824 M daltons (Da), rearrange and solve the above equation as follows:

1% = (

) * 10 = (1%) * (MW

molar

-1cm-1

and a molecular weight (MW) of 66,400

* 10) / (MW

molar

and 1%

)

protein

)

protein

1% = (43,824 * 10) / 66,400 Da) 1% = 6.6 g/100 mL

Conversions Between g/100 mL and mg/mL

Example: If measured absorbance for a protein sample at 280 nm relative to the reference is 5.8 A, protein concentration can be calculated as:

in mg/mL = (A / 1%) * 10

protein

= (A / 1%) * 10

= (5.8/6.6 g/100 mL) * 10

= 8.79 mg/mL

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5 Protein Applications

Measure Protein A280

Calculated protein concentrations are based on the absorbance value at 280 nm, the selected (or entered) extinction coefficient and the sample pathlength. A single-point baseline correction (or analysis correction) may be applied.

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on sample sequence.

Absorbance values at 260 nm and 280 nm are used to calculate purity ratios for the measured protein samples.

Purity ratios are sensitive to the presence of contaminants in the sample, such as residual solvents and reagents typically used during sample purification.

Measured Values

A280 absorbance

Note: For micro-volume absorbance measurements and

measurements taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

• Protein absorbance values are measured at 280 nm using the normalized spectrum. If Baseline Correction is not selected, this is the reported A280 value and the value used to calculate protein concentration.

• If Baseline Correction is selected, the normalized and baseline-corrected absorbance value at 280 nm is reported and used to calculate protein concentration.

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see

General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

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Protein Applications

Measure Protein A280

Reported Values

• Protein concentration. Reported in selected unit (mg/mL or µg/mL). Calculations are based on Beer-Lambert equation using corrected protein absorbance value.

• A260/A280 purity ratio. Ratio of corrected absorbance at 260 nm to corrected absorbance at 280 nm. An A260/A280 purity ratio of ~0.57 is generally accepted as “pure” for proteins.

Note: Although purity ratios are important indicators of sample quality, the best indicator of protein quality is functionality in the downstream application of interest (e.g., real-time PCR).

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5 Protein Applications

Measure Protein A205

Measures the concentration of purified protein populations that absorb at 205 nm.

Measure A205 Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Protein Concentration at A205

Use the Protein A205 application to quantify purified peptides and other proteins that contain peptide bonds, which exhibit absorbance at 205 nm. This application reports protein concentration and two absorbance values (A205 and A280). A single-point baseline correction can also be used. This application does not require a standard curve.

Note If your samples contain mainly amino acids such as tryptophan or tyrosine, or cys-cys disulfide bonds, use the Protein A280 application instead of Protein A205.

To measure Protein A205 samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Before you begin...

Pedestals.

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Protein Applications

Measure Protein A205

To measure a Protein A205 sample

1. From the Home screen, from the Proteins tab, select Protein A205.

2. Specify a sample type and baseline correction if desired.

3. Pipette 1–2 µL of the blanking solution onto the lower pedestal and lower the arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette light path with the instrument light path.

4. Tap Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the blanking cuvette.

6. Pipette 2 µL sample solution onto the pedestal and lower the arm, or insert the sample cuvette into the cuvette holder.

7. Start the sample measurement:

– Pedestal: If Auto-Measure is On, lower arm; if Auto-Measure is off, lower

arm and tap Measure.

– Cuvette: Tap Measure

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, tap End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

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Protein A205 Reported Results

Protein A205 measurement screen (Local Control)

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Here is an example:

tap to open

Sample name; tap to edit

UV spectrum

Protein concentration

Tap to select unitMenu of options;

Absorbance at

205 nm

Absorbance at

280 nm

Tap r ow to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Drag tab down/up

Pinch and zoom to adjust axes; double-tap to reset

Swipe screen left to view table with more measurement results

Tap to end experiment and export data

to see more/less sample data

Note Micro-volume absorbance measurements and measurements taken with

nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

Protein A205 reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

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Protein Applications

Measure Protein A205

Application

Baseline Correction wavelength

Sampling method

Baseline Correction absorbance

Sample name;

tap to edit

Date/time measured

Protein conc.

Absorbance at

205 nm

Absorbance at

280 nm

Sample type

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5 Protein Applications

Measure Protein A205

Protein A205 measurement screen (PC Control)

For each measured sample, this application shows the absorbance spectrum and reported results. Here is an example:

Click to select cuvette or pedestal measurement

Sample name; Click to edit

Portein A205 Setup

Run Blank

Measure sample

Click to end experiment and export data

Menu of options; click to open

Protein concentration

UV spectrum

Absorbance

values

Click and Drag to adjust axes; double-click to reset

Click to select unit

Click row to select sample and update spectrum; click more rows to overlay up to five spectra.

Related Topics

• Basic Instrument Operations

• Protein A205 Calculations

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Settings for Protein A205 Measurements

To show the Protein A205 settings, in local instrument control, from the Protein A205 measurement screen, tap > Protein A205 Setup.

From the PC control software, from the Protein A205 measurement screen, select the settings icon to view Protein A205 Setup.

Protein A205 settings

The Protein A205 application provides a variety of method options for protein analysis.

Protein Applications

Measure Protein A205

Mass Ext.

Setting Available Options

Sample type 31 31 Assumes 0.1% (1 mg/mL) at 205 nm = 31

Scopes 27 + 120 *

Coefficient (L/gm-cm)

(A280/A205)

Description

Assumes 120 * (A280/A205)

0.1% (1 mg/mL) at 205 nm = 27 +

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5 Protein Applications

Measure Protein A205

Setting Available Options

Mass Ext. Coefficient (L/gm-cm)

Description

Baseline Correction

Other protein

1%)

(

On or off

Enter baseline correction wavelength in nm or use default value (340 nm)

User entered mass extinction coefficient

N/A Corrects for any offset caused by light

Assumes protein has known mass extinction

coefficient (). Enter mass extinction

coefficient in L/gm-cm for 1 mg/mL ( protein solution.

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Baseline Correction.

0.1%)

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Calculations for Protein A205 Measurements

Protein Applications

Measure Protein A205

As with the other protein applications, Proteins A205 uses the Beer-Lambert

equation to correlate absorbance with

concentration based on the sample’s extinction coefficient and pathlength.

This application offers three options (shown at right) for selecting an appropriate extinction coefficient for each measured sample, to be used in conjunction with Beer’s Law to calculate sample concentration.

If the extinction coefficient of the sample is

known, choose the 1% (mass) option and

enter the value. Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution.

Tip: Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

Calculated protein concentrations are based on the absorbance value at 205 nm, the selected (or entered) extinction coefficient and the sample pathlength. A single-point baseline correction may also be applied.

Concentration is reported in mass units. Calculators are available on the Internet to convert concentration from mass to molar units based on the sample sequence.

Available Options for Extinction Coefficient

• 31, assumes

• Scopes, assumes

0.1% (1 mg/mL) at 205 nm = 31

0.1% (1 mg/mL) at 205 nm = 27 +

120 * (A280/A205)

• Other protein, enter mass extinction coefficient in

L/gm-cm for 1 mg/mL (

0.1%) protein solution

Note: See Sample Type for details.

Measured Values

A205 absorbance

Note: For micro-volume absorbance measurements and

measurements taken with nonstandard (other than 10 mm) cuvettes, the spectra are normalized to a 10 mm pathlength equivalent.

• Protein absorbance values are measured at 205 nm using the normalized spectrum. If Baseline Correction is not selected, this is the reported A205 value and the value used to calculate protein concentration.

• If Baseline Correction is selected, the normalized and baseline-corrected absorbance value at 205 nm is reported and used to calculate protein concentration.

A280 absorbance

• Normalized and baseline-corrected (if selected) absorbance value at 280 nm is also reported.

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5 Protein Applications

Measure Protein A205

Sample Pathlength

• For micro-volume measurements, the software selects the optimal pathlength (between 1.0 mm and 0.03 mm) based on sample absorbance at the analysis wavelength.

• For cuvette measurements, pathlength is determined by the cuvette Pathlength setting in the software (see

General Settings).

• Displayed spectra and absorbance values are normalized to a 10 mm pathlength equivalent.

Reported Values

• Protein concentration. Reported in selected unit (mg/mL or µg/mL). Calculations are based on Beer-Lambert equation using corrected protein absorbance value.

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Measure Proteins and Labels

Measures the concentration of purified proteins that have been labeled with up to two fluorescent dyes.

Measure Labeled Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Labeled Protein Samples

Protein Applications

Measure Proteins and Labels

Use the Proteins and Labels application to quantify proteins and fluorescent dyes for protein array conjugates, as well as metalloproteins such as hemoglobin, using wavelength ratios. This application reports protein concentration measured at 280 nm, an A269/A280 absorbance ratio, and the concentrations and measured absorbance values of the dyes, allowing detection of dye concentrations as low as

0.2 picomole per microliter. This information is useful for evaluating protein/dye conjugation (degree of labeling) for use in downstream applications.

To measure labeled protein samples

NOTICE

• Do not use a squirt or spray bottle on or near the instrument as liquids will flow into the instrument and may cause permanent damage.

• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently damage the quartz fiber optic cables.

Before you begin...

Pedestals.

To measure a labeled protein sample

1. From the Home screen, select the Proteins tab and then select Protein & Labels.

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5 Protein Applications

Measure Proteins and Labels

2. Specify the sample type and the type of dye(s) used.

3. Pipette 1–2 µL of the blanking solution onto the lower pedestal and lower the

4. Tap Blank and wait for the measurement to complete.

5. Lift the arm and clean both pedestals with a new laboratory wipe, or remove the

6. Pipette 2 µL sample solution onto the pedestal and lower the arm, or insert the

Tip: Select a dye from the pre-defined list or add a custom dye using the

Dye/Chromophore Editor.

arm, or insert the blanking cuvette into the cuvette holder.

Tip: If using a cuvette, make sure to align the cuvette light path with the instrument light path.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you lower the arm. (This option is not available for cuvette measurements.)

blanking cuvette.

sample cuvette into the cuvette holder.

7. Start the sample measurement:

– Pedestal: If Auto-Measure is On, lower arm; if Auto-Measure is off, lower arm

and select Measure.

– Cuvette: select Measure

When the sample measurement is completed, the spectrum and reported values are displayed (see the next section).

8. When you are finished measuring samples, select End Experiment.

9. Lift the arm and clean both pedestals with a new wipe, or remove the sample cuvette.

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Protein Applications

Measure Proteins and Labels

Peptide backbone

Dye absorbance peak used to calculate dye concentration

A280 absorbance peak used to calculate protein concentration

Typical sample spectrum measured with Proteins & Labels

Related Topics

• Best practices for protein measurements

• Measure a Micro-Volume Sample

• Measure a Sample Using a Cuvette

• Prepare Samples and Blanks

• Basic Instrument Operations

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Measure Proteins and Labels

Proteins & Labels Reported Results

Proteins & Labels measurement screen

For each measured sample, this application shows the absorbance spectrum and a summary of the results. Below is an example of the measurement screen of the PC control software:

Application

Sampling method

Sample name of next measurement; select to edit

UV spectrum

Run Blank

Proteins &

Labels setup

Measure sample

End experiment

Menu of options; click to open

Right click graph area to view display

options

Menu of table options; click to choose which columns to report

Sample name; select to edit

Protein concentration

Dye concentration(s)

Click row to select sample and update spectrum.

Measurement screen of PC Control software

Note

• A baseline correction is performed at 850 nm (absorbance value at 850 nm is subtracted from absorbance values at all wavelengths in sample spectrum).

• Micro-volume absorbance measurements and measurements taken with nonstandard cuvettes are normalized to a 10.0 mm pathlength equivalent.

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Protein Applications

Measure Proteins and Labels

Menu of options; tap to open

UV-visible spectrum

Sample name; tap to edit

Protein concentration

Tap to select unit

Dye concentration(s)

Tap r ow to select sample and update spectrum; tap more rows to overlay up to five spectra. Press and hold sample row to view measurement details.

Pinch and zoom to adjust axes; double-tap to reset

Swipe screen left to view table with more measurement results

Measurement screen of NanoDrop One local control

Proteins & Labels reported values

The initial screen that appears after each measurement (see previous image) shows a summary of the reported values. To view all reported values, press and hold the sample row. Here is an example:

Reported values for Proteins & Labels application

• Sample details (application and sampling method used, i.e., pedestal or cuvette)

• Sample Name

• Creation date

• Protein

Drag tab down/up to see more/less

sample data Tap to end experiment and export data

• A280

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5 Protein Applications

Measure Proteins and Labels

• Sample Type

• Dye 1/Dye 2

• Sloping Dye Correction

• Analysis Correction

Related Topics

• Basic Instrument Operations

• Proteins & Labels calculations

Settings for Proteins and Labels Measurements

To show the Proteins & Labels settings, from the Proteins & Labels measurement screen, tap > Proteins & Labels Setup.

Setting Available Options

Sample

type

1 Abs = 1 mg/mL

BSA

IgG

Lysozyme

Other protein

 + MW)

(

Other protein

1%)

(

Mass Ext. Coefficient (L/gm-cm)

General reference

6.7

13.7

26.4

user-entered molar extinction coefficient/ molecular weight

User entered mass extinction coefficient

Description

Tap here for detailed description of each

available setting.

Each sample type applies a unique extinction coefficient to the protein calculations. If the extinction coefficient of the sample is known, choose the option and enter the value. Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution. If you only need a rough estimate of protein concentration and the sample extinction coefficient is unknown, select the 1 Abs=1 mg/mL sample type option.

Tip: Ideally, the extinction coefficient should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

 + MW (molar) or 1% (mass)

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Setting Available Options

Analysis Correction

On or off

Enter analysis correction wavelength in nm or use default value (340 nm)

Protein Applications

Measure Proteins and Labels

Mass Ext. Coefficient (L/gm-cm)

Description

N/A Corrects sample absorbance measurement

for any offset caused by light scattering particulates by subtracting absorbance value at specified analysis correction wavelength from absorbance value at analysis wavelength. Corrected value is used to calculate sample concentration.

Tip: If the sample has a modification that absorbs light at 340 nm, select a different correction wavelength or turn off Analysis Correction.

Dye 1/Dye 2

Type

Cy3, 5, 3.5, or 5.5, Alexa Fluor 488, 546, 555, 594, 647, or 660

See

Dye/Chromophore Editor for specific

values for each dye

Dye 1/Dye 2 Unit

picomoles/microliter (pmol/uL), micromoles

not applicable Select unit for reporting dye concentrations.

(uM), or millimoles (mM)

Sloping Dye Correction

To add or edit a custom protein, use Protein Editor.

Analysis Correction affects calculation for protein concentration only.

To add custom dye or edit list of available dyes, use Dye/Chromophore Editor.

Sloping Dye Correction affects calculations for dye concentration only.

On or off Corrects dye absorbance measurements for

Select pre-defined dye used to label sample material, or one that has been added using Dye/Chrom. Editor.

any offset caused by light scattering particulates by subtracting absorbance value of a sloping baseline from 400 nm to 850 nm from absorbance value at dye’s analysis wavelength.

Related Topics

• Instrument Settings

• Protein Editor

• Dye/Chromophore Editor

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Detection Limits for Proteins and Labels Measurements

Detection limits and reproducibility specifications for purified BSA proteins and dyes that are pre-defined in the software are provided here. The BSA lower detection limit and reproducibility values apply to any protein sample type. The upper detection limits are dependent on the upper absorbance limit of the instrument and the sample’s extinction coefficient.

To calculate upper detection limits for other (non-BSA) protein sample types

To calculate upper detection limits in mg/mL for proteins, use the following equation:

(upper absorbance limit

instrument

/mass extinction coefficient

For example, if the sample’s mass extinction coefficient at 280 nm is 6.7 for a 1% (10 mg/mL) solution, the equation looks like this:

(550 / 6.7) * 10 = 824.6 (or ~825)

Related Topics

• Detection Limits for All Applications

Calculations for Proteins and Labels Measurements

As with the other protein applications, Proteins & Labels uses the Beer-Lambert

equation to correlate absorbance with

concentration based on the sample’s extinction coefficient and pathlength.

Available Options for Extinction Coefficient

• 1 Abs = 1 mg/mL, where sample type and/or ext.

coefficient is unknown (produces rough estimate of protein concentration)

• BSA (Bovine Serum Albumin, 6.7 L/gm-cm)

• IgG (any mammalian antibody, 13.7 L/gm-cm)

• Lysozyme (egg white lysozyme, 26.4 L/gm-cm)

sample

) * 10

If the extinction coefficient of the sample is

known, choose the  + MW (molar) or

1% (mass) option and enter the value.

Otherwise, calculate the extinction coefficient or choose the option that best matches the sample solution.

• Other protein (

coefficient

• Other protein (

coefficient

 + MW), user-specified molar ext.

1%), user-specified mass ext.

Note: See Sample Type for details.

Tip: Ideally, the extinction coefficient

should be determined empirically using a solution of the study protein at a known concentration using the same buffer.

100 NanoDrop One User Guide Thermo Scientific

Thermo Fisher Scientific NanoDrop One User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Contents

About the Spectrophotometer

Features

Touchscreen

Cuvette Holder

USB-A port

Accessories

DYMO™ LabelWriter™ 450 USB Label Printer

PR-1 Pedestal Reconditioning Kit

PV-1 Performance Verification Solution

Instrument Detection Limits

Instrument Set up

Register Your Instrument

Update Software

Setting Up User Account Control (Optional)

User Account Control

Security Administration Policies

Technical Support

For U.S./Canada Support, please contact:

For International Support, please contact:

Application Measurement Ranges

Detection Limits for All Applications

Detection limits for standard applications

Detection limits for pre-defined dyes

Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

Measure dsDNA, ssDNA or RNA

Best practices for nucleic acid measurements

Nucleic Acid Reported Results

Settings for Nucleic Acid Measurements

Calculations for Nucleic Acid Measurements

Measure Microarray

Measure Microarray Samples

Microarray Reported Results

Settings for Microarray Measurements

Calculations for Microarray Measurements

Measure using a Custom Factor

Measure Nucleic Acid using a Custom Factor

Custom Factor Reported Results

Settings for Nucleic Acid Measurements using a Custom Factor

Detection Limits for Nucleic Acid Measurements using a Custom Factor

Measure Oligo DNA or Oligo RNA

Measure Oligo DNA or Oligo RNA

Oligo Reported Results

Settings for Oligo DNA and Oligo RNA Measurements

Detection Limits for Oligo DNA and Oligo RNA Measurements

Calculations for Oligo DNA and Oligo RNA Measurements

Protein Applications

Measure Protein A280

Measure Protein Concentration at A280

Best practices for protein measurements

Protein A280 Reported Results

Settings for Protein A280 Measurements

Protein editor

Detection Limits for Protein A280 Measurements

Calculations for Protein A280 Measurements

Measure Protein A205

Measure Protein Concentration at A205

Protein A205 Reported Results

Settings for Protein A205 Measurements

Calculations for Protein A205 Measurements

Measure Proteins and Labels

Measure Labeled Protein Samples

Proteins & Labels Reported Results

Settings for Proteins and Labels Measurements

Detection Limits for Proteins and Labels Measurements

Calculations for Proteins and Labels Measurements