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Juno

Fluidigm Juno Getting Started Manual

Genotyping with Juno
Getting Started Guide
PN 100-7074 D1
For Research Use Only. Not for use in diagnostic procedures.
Information in this publication is subject to change without notice. It is Fluidigm policy to improve products as new techniques and components become available. Therefore, Fluidigm reserves the right to change specifications at any time. Every effort has been made to avoid errors in the text, diagrams, illustrations, figures, and screen captures. However, Fluidigm assumes no responsibility for any errors or omissions. In no event shall Fluidigm be liable for any damages in connection with or arising from the use of this publication.
Patent and Limited License Information
Fluidigm products are covered by issued and pending patents in the United States and other countries. Patent and limited license information is available at fluidigm.com/legalnotices
Limited Use License to Perform Preamplification with Fluidigm IFCs
A license to use Thermo Fisher Scientific’s patented preamplification method workflows involving a Fluidigm integrated fluidic circuit (IFC) can be obtained (i) with purchase of a Fluidigm IFC from Fluidigm Corporation or (ii) by a separate license from Thermo Fisher Scientific. For licensing information, contact outlicensing@lifetech.com.
Limited Digital PCR License
A license to use Thermo Fisher Scientific’s patented digital PCR method in all fields other than in the Sequencing Field, the Mass Spectrometry Field, and the Prenatal Field in workflows involving a Fluidigm IFC can be obtained (i) with purchase of a Fluidigm IFC from Fluidigm Corporation or (ii) by a separate license from Thermo Fisher Scientific. For licensing information, contact outlicensing@lifetech.com.
Trademarks
Fluidigm, the Fluidigm logo, Biomark, EP1, Juno, and SNP Type are trademarks or registered trademarks of Fluidigm Corporation in the United States and/or other countries. All other trademarks are the sole property of their respective owners.
For EU's WEEE directive information, go to fluidigm.com/compliance
© 2017 Fluidigm Corporation. All rights reserved. 09/2017
For technical support visit fluidigm.com/support.
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Genotyping with Juno Getting Started Guide
Contents
About This Guide 4
Purpose 4
How to Use This Guide 5
Safety Alert Conventions 5
Safety Alerts for Chemicals 5
Safety Alerts for Instruments 5
Safety Data Sheets 6
Getting Started 7
Workflow 7
Best Practices 7
Related Documentation 7
Chapter 1: Product Information 8
Required Kit Contents 8
TaqMan Assay Kit 8
SNP Type Assay Kit 9
Required Reagents 10
TaqMan Assays 10
SNP Type Assays 10
Suggested Reagents 10
Required Consumables 11
Required Equipment 11
Obtain the Minimum Required Genomic DNA 14
Prepare the Sample Mix 15
Load and Run the IFC on Juno 16
Perform Genotyping Analysis on the Samples 19
19
Chapter 3: Genotyping with the Juno
96.96 Genotyping IFC Using SNP Type Assays 20
Prepare Assay and Sample Mixes 20
Prepare the 200 nM Primer Pool for Preamplification 20
Prepare 2X SNP Type Assays 21
Prepare the Assay Mix 22
Obtain the Minimum Required Genomic DNA 22
Prepare the Sample Mix 23
Load and Run the IFC on Juno 24
Perform Genotyping Analysis on the Samples 27
27
Appendix A: Suggested Kits 28
TaqMan Assay Kit 28
SNP Type Assay Kit 28
Suggested Reagents to Use with TaqMan Assay and SNP Type Assay Kits 29
Suggested Equipment 11
Required Software 12
IFC Type and Related Scripts 12
Chapter 2: Genotyping with the Juno 96.96 Genotyping IFC Using TaqMan Assays 13
Prepare Assay and Sample Mixes 13
Prepare the Primer Pool for Preamplification 13
Prepare 2X TaqMan Assays for Genotyping 14
Prepare the Assay Mix 14
Genotyping with Juno Getting Started Guide
Appendix B: Safety 30
General Safety 30
Instrument Safety 30
Electrical Safety 31
Chemical Safety 31
Disposal of Products 31
3
About This Guide
CAUTION ABBREVIATED SAFETY ALERTS. Hazard symbols and hazard
types specified in procedures may be abbreviated in this document. For complete safety information, see the safety appendix on page 30.
Purpose
This guide describes how to perform genotyping of low-concentration DNA with the Juno™ 96.96 Genotyping IFC (integrated fluidic circuit) on the Juno™ system. This is possible through advanced microfluidics technology that integrates preamplification and genotyping reactions of up to 96 samples and 96 genotyping assays in a single workflow on an IFC.
The IFC produces 9,216 genotypes in less than three hours using a simple workflow with minimal hands-on time. Samples are loaded into individual inlets of the Juno 96.96 Genotyping IFC, then distributed across multiple reaction chambers in nanoliter-volume aliquots. With high-quality samples, detecting the specific targets requires thermal cycling for preamplification and PCR for genotyping on the instrument.
After genotyping is performed on the Juno system, the IFC is scanned on the EP1™ system or the Biomark™ HD system to collect genotyping data for later analysis.
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Genotyping with Juno Getting Started Guide
How to Use This Guide
The chapters in this guide are organized according to assay type. Refer to the appropriate chapter to run the Juno 96.96 Genotyping IFC on the Juno system.
For detailed instructions on instrument and software operation, refer to the Juno System User Guide (PN 100-7070).
Safety Alert Conventions
This guide uses specific conventions for presenting information that may require your attention. Refer to the following safety alert conventions.
Safety Alerts for Chemicals
Fluidigm follows the United Nations Globally Harmonized System (GHS) for communicating chemical hazard information. GHS provides a common means of classifying chemical hazards and a standardized approach to chemical label elements and safety data sheets (SDSs). Key elements include:
About This Guide
How to Use This Guide
• Pictograms that consist of a symbol on a white background within a red diamond­shaped frame. Refer to the individual SDS for the applicable pictograms and warnings pertaining to the chemicals being used.
• Signal words that alert the user to a potential hazard and indicate the severity level. The signal words used for chemical hazards under GHS:
DANGER Indicates more severe hazards.
WARNING Indicates less severe hazards.
Safety Alerts for Instruments
For hazards associated with instruments, this guide uses the following indicators:
• Pictograms that consist of a symbol on a white background within a black triangle­shaped frame.
Genotyping with Juno Getting Started Guide
5
About This Guide
Safety Data Sheets
• Signal words that alert the user to a potential hazard and indicate the severity level. The signal words used for instrument hazards:
DANGER Indicates an imminent hazard that will result in severe injury or death if not avoided.
WARNING Indicates a potentially hazardous situation that could result in serious injury or death.
CAUTION Indicates a potentially hazardous situation that could result in minor or moderate personal injury.
IMPORTANT Indicates information necessary for proper use of products or successful outcome of experiments.
Safety Data Sheets
Read and understand the SDSs before handling chemicals. To obtain SDSs for chemicals ordered from Fluidigm Corporation, either alone or as part of this system, go to fluidigm.com/sds and search for the SDS using either the product name or the part number.
Some chemicals referred to in this user guide may not have been provided with your system. Obtain the SDSs for chemicals provided by other manufacturers from those manufacturers.
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Genotyping with Juno Getting Started Guide
Getting Started
Workflow
Reagent Handling Automated Steps Estimated Times
1 Prepare preamplification and
genotyping assay and sample mixes
2 Pipet preamplification, genotyping
mixes, and control line fluid into the IFC
3
4
Run a script to preamplify and genotype the DNA
Perform genotyping analysis on EP1 or Biomark systems
30–60 minutes
10–20 minutes
2.5 hours (TaqMan protocol); 3.5 hours (SNP Type protocol)
5–10 minutes
Best Practices
• Use good laboratory practices to minimize contamination of samples. Use a new
pipette tip for every new sample. Whenever possible, separate pre- and post­PCR activities. Dedicate laboratory materials to designated areas.
• Unless otherwise specified, thaw reagents at room temperature, then use them at room temperature. Store reagents at their specified storage temperatures. (See “Required Kit Contents” on page 8.)
• Vortex reagents for 20 seconds, and then centrifuge reagents for 2 seconds before use.
Related Documentation
Go to fluidigm.com/documents
Genotyping with Juno Getting Started Guide
7
Chapter 1: Product Information
Required Kit Contents
The kits include the reagents required for preparing 10 IFCs to use on the Juno system. For suggested kits, see “Suggested Kits” on page 28.
IMPORTANT
• Do not pipet reagents from the TaqMan and SNPType assay kits into the same IFC.
Use a different IFC for each kit. Do not mix reagents from different kits.
• Unless otherwise specified, thaw reagents at room temperature, then use them at
room temperature. Store reagents at their specified storage temperatures. Vortex reagents for 20 seconds, then centrifuge reagents for 2 seconds before use.
TaqMan Assay Kit
Box Component Cap Color Quantity Volume
per Tube (mL)
Juno Genotyping Kit for 10 IFCs (PN 100-8362)
Juno GT Flux Fluid
Juno GT Preamp Master Mix
Dilution Reagent Natural 2 tubes 1.7
Probe GT Master Mix Gold 2 tubes 1.6
Juno GT Flux Fluid Purple 1 tube 0.9
Juno 96.96 Genotyping IFC— 10 IFCs
Juno 96.96 GT Control Line Fluid
Light purple
— 10 IFCs —
— 2 boxes;
Probe GT Master Mix
Juno GT Preamp Master Mix
Dilution Reagent
1 tube 1.35
20 syringes/ box
—
Storage
–20 ºC
Room temperature
Figure 1. Juno™ Genotyping Kit for 10 IFCs (PN 100-8362).
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Genotyping with Juno Getting Started Guide
SNP Type Assay Kit
Chapter 1: Product Information
Required Kit Contents
Box Component Cap Color Quantity Volume
per Tube or Bottle
Juno SNP Type Genotyping Kit for 10 IFCs (PN 100-8364)
Juno GT Preamp Master Mix
Juno SNP Type GT Master Mix
60X SNP Type Reagent Amber
Light
1 tube 1.35 mL
purple
Light
2 tubes 1.6 mL
blue
2 tubes 70 μL
Juno GT Flux Fluid Purple 1 tube 1.0 mL
Dilution Reagent Natural • 2 bottles
• 1 tube
Juno 96.96 Genotyping
— 10 IFCs —
• 3.7 mL
• 1.7 mL
IFC—10 IFCs
Juno 96.96 GT Control Line Fluid
— 2 boxes;
20 syringes/
—
box
Juno GT Flux Fluid
60X SNP Type Reagent
Storage
–20 ºC
Room temperature
Dilution Reagent
Juno SNP Type GT Master Mix
Juno GT Preamp Master Mix
Figure 2. Juno™ SNP Type Genotyping Kit for 10 IFCs (PN 100-8364).
Genotyping with Juno Getting Started Guide
9
Chapter 1: Product Information
Required Reagents
Required Reagents
TaqMan Assays
Product Name Company Part Number
20X, 40X, or 80X TaqMan assays
®
genotyping
Thermo Fisher Scientific
—
SNP Type Assays
Product Name Company Part Number
SNP Type assays specific target amplification Primers (100 μM STA)
SNP Type assays ASP1/ASP2 (100 μM each)
SNP Type assays LSP (100 μM each)
Fluidigm —
Fluidigm —
Fluidigm —
Suggested Reagents
Product Name Company Part Number
UltraPure Water
™
DNase/RNase-Free Distilled
Thermo Fisher Scientific
10977-015
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Genotyping with Juno Getting Started Guide
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