Eppendorf BioSpectrometer fluorescence User Manual

nualoSpectrometer® fluorescence

www.eppendorf.com/myeppendorf

manual

Eppendorf BioSpectrometer® fluorescence

Operating manual

Trademarks

Eppendorf

and the Eppendorf logo, Eppendorf BioSpectrometer®, Eppendorf SpectraZoom®, and UVette®

are registered trademarks of Eppendorf AG, Hamburg, Germany.

is a registered trademark of GE Healthcare UK Ltd., Buckinghamshire, UK.

Hellma

OliGreen

is a registered trademark of Hellma GmbH & Co. KG, Müllheim, Germany.

, PicoGreen®, RiboGreen®, NanoOrange® and Qubit® are registered trademarks of Molecular

Probes, Inc., Eugene, OR, USA.

Trademarks are not marked in all cases with ™ or

in this manual.

This product is manufactured under license to issued U.S. Patent No. 6,122,052.

Protected by U.S. Patent No. 8,464,171.

6137 900.058-02/052014

Table of contents

Eppendorf BioSpectrometer

fluorescence

English (EN)

Table of contents

1 Operating instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.1 Using this manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2 Danger symbols and danger levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2.1 Danger symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2.2 Danger levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.3 Symbols used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.4 Abbreviations used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2 Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.1 Main illustration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.2 Delivery package. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.3 Features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.3.1 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.3.2 Operation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.3.3 Result output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.3.4 Device self test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3 Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.1 Intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.2 User profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.3 Warnings for intended use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.3.1 Personal injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.3.2 Damage to device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.4 Information on product liability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.5 Safety instructions located on the device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4 Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.1 Preparing installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.2 Selecting the location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.3 Connecting the device to the mains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.4 Connecting the printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.4.1 Thermal printer DPU-S445 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.4.2 Thermal printer DPU-414 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.5 Connecting PC or USB stick for data export. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5 Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.1 Overview of operating controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.1.1 Entering text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.2 Inserting the cuvette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5.3 Summary of the measuring procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

5.3.1 Preparing the measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

5.3.2 Measuring procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

5.3.3 Important measurement instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Table of contents

Eppendorf BioSpectrometer

fluorescence

English (EN)

6 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

6.1 Selecting a method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

6.2 Photometry method description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

6.2.1 Absorbance method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

6.2.2 Routine method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

6.2.3 Basic method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

6.2.4 Advanced method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

6.3 Fluorimetry method description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

6.3.1 Routine method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

6.3.2 Basic method group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

6.4 Method parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

6.5 Method procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

6.5.1 Check parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

6.5.2 Measure standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

6.5.3 Measure samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

6.5.4 Measure samples: Results displays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

6.5.5 Process results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

6.5.6 Process results: Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

6.5.7 Print & export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

6.5.8 Finish the series of measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

7 Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

7.1 Functions of the User main group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

7.1.1 Results memory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

7.1.2 General method parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

7.1.3 Absorbance spectra library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

7.1.4 Device settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

7.1.5 Device calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

7.1.6 Info . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

8 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

8.1 Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

8.1.1 Cleaning the cuvette shaft cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

8.2 Disinfection/Decontamination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

8.3 Checking the device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

8.3.1 Checking the spectrometer unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

8.3.2 Checking the fluorescence unit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

8.3.3 Device self-test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

8.4 Replacing fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

8.5 Decontamination before shipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

9 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

9.1 General errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

9.2 Error messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

9.3 Result flags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

10 Transport, storage and disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

10.1 Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

10.2 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

10.3 Disposal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Table of contents

Eppendorf BioSpectrometer

fluorescence

English (EN)

11 Technical data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

11.1 Power supply. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

11.2 Ambient conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

11.3 Weight/dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

11.4 Photometric properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

11.5 Fluorimeter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

11.6 Further technical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

11.7 Application parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

12 Evaluation procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

12.1 Absorbance values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

12.1.1 Blank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

12.1.2 Background correction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

12.1.3 Cuvette correction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

12.2 Evaluation with factor or standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

12.3 Evaluation with standard curve/line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

12.4 Dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

12.5 Special evaluation procedures for nucleic acids and protein UV . . . . . . . . . . . . . . . . . . . . . . . . 90

12.5.1 Correction A

and correction A

260

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

280

12.5.2 Ratios A260/A280 and A260/A230. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

12.5.3 Conversion to molar concentrations and nucleic acid quantities . . . . . . . . . . . . . . . . . 92

12.5.4 Calculating the factor for protein in "General Method Parameter" . . . . . . . . . . . . . . . 93

12.6 Special evaluation procedures for the dye methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

12.6.1 Calculating the factor for the dye from the absorbance coefficient . . . . . . . . . . . . . . . 94

12.6.2 Calculation of the FOI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

12.6.3 Conversion to amounts of dye. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

12.7 Dual wavelength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

12.8 Fluorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

12.8.1 RFU values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

12.8.2 Blank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

12.8.3 Evaluation with standard and standard curve/line, dilution . . . . . . . . . . . . . . . . . . . . . 96

13 Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Certificates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Table of contents

Eppendorf BioSpectrometer

English (EN)

fluorescence

Operating instructions

Eppendorf BioSpectrometer

fluorescence

English (EN)

1 Operating instructions

1.1 Using this manual

 Read this operating manual completely before using the device for the first time. Also observe the

instructions for use of the accessories.

 This operating manual is part of the product. Thus, it must always be easily accessible.

 Enclose this operating manual when transferring the device to third parties.

 You will find the current version of the operating manual for all available languages on our webpage

under www.eppendorf.com

1.2 Danger symbols and danger levels

The safety instructions of this operating manual indicate the following danger symbols and danger levels:

1.2.1 Danger symbols

Electric shock Explosion

Toxic substances Hazard point

Material damage

1.2.2 Danger levels

DANGER Will lead to severe injuries or death.

WARN ING May lead to severe injuries or death.

CAUTION May lead to light to moderate injuries.

NOTICE May lead to material damage.

Operating instructions

Eppendorf BioSpectrometer

English (EN)

1.3 Symbols used

Depiction Meaning

 Actions without a specified order

• List

or sample

or [Copy]

fluorescence

Actions in the specified order

Press this key to perform the described action.

Press this softkey to perform the described action.

Additional information

Operating instructions

Eppendorf BioSpectrometer

fluorescence

English (EN)

1.4 Abbreviations used

Absorbance

DNA

Deoxyribonucleic acid

dsDNA

Double-stranded DNA

Dye methods Methods for dye labels group for measuring dyed biomolecules

FOI

Frequency of Incorporation: measure for the amount of dye molecules in relation to the amount of nucleotides in dyed biomolecules

mol/L (molar)

OD600

Optical density at a wavelength of 600 nm

RFU

Relative Fluorescence Unit: measure for the intensity in fluorescence measurements

RNA

Ribonucleic acid

ssDNA

Single-stranded DNA

Ultraviolet radiation

Vis

Visible light

Coefficient of variation (standard deviation/average value) in percent

Operating instructions

Eppendorf BioSpectrometer English (EN)

fluorescence

2 Product description

2.1 Main illustration

Abb. 2-1: Front and rear view

Product description

Eppendorf BioSpectrometer

fluorescence

English (EN)

abc

def

method

ghi

jkl

mno

function

pqrs

tuv

wxyz

µ %

exit

delete

standard

enter

Fig. 2-1: Front and rear view

1Display

2Cuvette shaft

2 3

absorbance

height

8.5 mm

blank

sample

absorbance

6Fuse holder

7 Mains/power connection

3Cuvette shaft cover

4 USB port for USB stick and printer

5 Mains/power switch

8USB port for PC

9 Connection for RS-232 printer

10 Operating controls

The name plate is located at the rear left on the bottom of the device.

2.2 Delivery package

Quantity Description

1 BioSpectrometer fluorescence

1 Power cord

4 4 UVettes

Original Eppendorf plastic cuvette, individually packaged, PCR clean, protein-free

1 Operating manual, in multiple languages

Product description

Eppendorf BioSpectrometer English (EN)

fluorescence

2.3 Features

The BioSpectrometer fluorescence combines two spectroscopic measuring procedures: spectrophotometry and fluorimetry. It is able to carry out both spectrophotometric measurements in the UV/Vis range of 200 nm to 830 nm and fluorimetric measurements at two defined wavelength combinations in the visible range (470 nm excitation/520 nm emission and 470 nm excitation/560 nm emission). It is intended for the measurement of liquids in cuvettes in development and research in the fields of molecular biology, biotechnology, biochemistry and cell biology. You can use glass and plastic cuvettes in a volume range of 1 μL to 3000 μL (photometry) or 60 μL to 3000 μL (fluorimetry).

2.3.1 Methods

Photometry

Numerous methods for concentration determination of nucleic acids, proteins, and dye-marked nucleic acids and proteins, and the OD 600 method for determining bacterial density via turbidity measurement, are already preprogrammed. Furthermore, method templates for various measurement and evaluation procedures (single and multiple wavelength measurements, taking spectra, evaluations with factor, standard and standard curve) are preprogrammed. It is possible to create individual methods on the basis of the preprogrammed methods and templates. The templates in the Absorbance method group can be used to quickly measure absorbances or spectra without an additional evaluation.

Fluorimetry

Methods for the concentration determination of nucleic acids with PicoGreen, RiboGreen, OliGreen and Qubit reagents, and the concentration determination of proteins with NanoOrange, are preprogrammed. For quick measurements with only two standards, short versions of nucleic acid methods are also included. As in spectrophotometry, method templates are preprogrammed for various evaluation procedures (using factor, standard, and standard curve).

2.3.2 Operation

The preprogrammed methods and templates are combined into clearly arranged groups from which the desired method can be quickly selected. After calling up the method, you are guided through the measuring procedure in clear steps. A help box in the display provides hints upon request. The 3 round measuring keys (standard, blank, sample) allow users to quickly start a measurement.

2.3.3 Result output

The BioSpectrometer fluorescence outputs the results using the device display and a printer available from Eppendorf. With a USB connection, you can transfer result data from the device to a USB stick, a printer or directly to a PC.

2.3.4 Device self test

The device automatically tests the function of the spectrometer unit and the fluorescence unit right after it has been switched on. Access the Device calibration function for a more comprehensive test (see Device self-test on p. 71).

Safety

fluorescence

English (EN)

Eppendorf BioSpectrometer

3Safety

3.1 Intended use

The BioSpectrometer fluorescence is to be used in molecular biology, biochemistry and cell biology research laboratories. The BioSpectrometer fluorescence is exclusively intended for use indoors. All country-specific safety requirements for operating electrical equipment in the laboratory must be observed.

The BioSpectrometer fluorescence is used for photometric concentration determination of analytes in liquids and recording of absorbance wavelength spectra in cuvettes. In addition, fluorescence measurements can be used to quantify biomolecules.

Only use Eppendorf accessories or accessories recommended by Eppendorf.

3.2 User profile

The device and accessories may only be operated by trained and skilled personnel.

Before using the device, read the operating manual carefully and familiarize yourself with the device's mode of operation.

3.3 Warnings for intended use

3.3.1 Personal injury

DANGER! Electric shock as a result of penetration of liquid.

 Switch off the device and disconnect the power plug before starting cleaning or

disinfection work.

 Do not allow any liquids to penetrate the inside of the housing.  Do not spray clean/spray disinfect the housing.  Only plug the device back in if it is completely dry, both inside and outside.

DANGER! Risk of explosion.

 Do not operate the device in areas where work is completed with explosive substances.  Do not use this device to process any explosive or highly reactive substances.  Do not use this device for processing any substances which could generate an explosive

atmosphere.

Safety

Eppendorf BioSpectrometer English (EN)

WARNING! Electric shock due to damage to device or mains cable.

 Only switch on the device if the device and mains cable are undamaged.  Only use devices that have been properly installed or repaired.  In case of danger, disconnect the device from the mains supply by pulling the power plug

from the device or the mains socket or, by using the isolating device intended for this purpose (e.g., emergency stop switch in the laboratory).

WARNING! Damage due to UV radiation.

Microliter cuvettes, e.g., Hellma® TrayCell (or microliter cuvettes with a similar design) divert the radiation from the light source within the cuvette so the radiation can escape upward when the lid is not closed.

 Before starting a measurement, ensure that the lid on the microliter cuvette is not open.

WARNING! Damage to health from toxic, radioactive or aggressive chemicals as well as infectious liquids and pathogenic germs.

fluorescence

 Observe the national regulations for handling these substances, the biological security

level of your laboratory, the material safety data sheets and the manufacturer's application notes.

 Wear personal protective equipment.  For comprehensive regulations about handling germs or biological material of risk group II

or higher, please refer to the "Laboratory Biosafety Manual" (source: World Health Organization, Laboratory Biosafety Manual, in its respectively current valid version).

WARNING! Damage to health due to contaminated device and accessories.

 Decontaminate the device and the accessories before storage and shipping.

CAUTION! Poor safety due to incorrect accessories and spare parts.

The use of accessories and spare parts other than those recommended by Eppendorf may impair the safety, functioning and precision of the device. Eppendorf cannot be held liable or accept any liability for damage resulting from the use of incorrect or non-recommended accessories and spare parts, or from the improper use of such equipment.

 Only use accessories and original spare parts recommended by Eppendorf.

3.3.2 Damage to device

NOTICE! Damage from the use of aggressive chemicals.

 Do not use any aggressive chemicals on the device or its accessories, such as strong and

weak bases, strong acids, acetone, formaldehyde, halogenated hydrocarbons or phenol.

 If the device has been contaminated by aggressive chemicals, immediately clean it by

means of a mild cleaning agent.

NOTICE! Damage to the device from fumigating with aggressive chemicals.

 Do not use fumigation to disinfect the device.

NOTICE! Corrosion from aggressive cleaning agents and disinfectants.

 Do not use corrosive cleaning agents, aggressive solvents or abrasive polishes.  Do not incubate the accessories in aggressive cleaning agents or disinfectants for a longer

period of time.

Eppendorf BioSpectrometer

Safety

fluorescence

English (EN)

NOTICE! Damage to electronic components due to condensation.

Condensate can form in the device after it has been moved from a cool environment to a warmer environment.

 After installing the device, wait at least for 3 h. Only then connect the device to the mains.

NOTICE! Function impairment due to mechanical damage.

 After mechanical damage to the device, ensure that the measuring and evaluation

functions of the device are operating correctly by completing an inspection.

NOTICE! Damage from overheating.

 Do not install the device near to any heat sources (e.g., heating, drying cabinet).  Do not expose the device to direct sunlight.  Ensure unobstructed air circulation. Keep free a clearance of at least 5 cm around all

ventilation grilles.

NOTICE! Material damage from incorrect use.

 Only use the product for its intended purpose as described in the operating manual.  Ensure adequate material resistance when using chemical substances.  In case of doubt, contact the manufacturer of this product.

NOTICE! Damage as a result of incorrect packing.

Eppendorf AG is not liable for damage caused by improper packing.

 The device may only be stored and transported in its original packaging.

Gerät nach dem Öffnen

justieren!

Adjust device after

opening!

Safety

Eppendorf BioSpectrometer

fluorescence

English (EN)

NOTICE! Damage due to improper cleaning of the cuvette shaft.

 Only clean the cuvette shaft using a moist cotton swab (see Cleaning on p. 65).  Do not allow any liquid to enter the cuvette shaft.  Do not reach with your fingers into the cuvette shaft.

3.4 Information on product liability

In the following cases, the designated protection of the device may be compromised. Liability for any resulting property damage or personal injury is then transferred to the operator:

• The device is not used in accordance with the operating manual.

• The device is used outside of its intended use.

• The device is used with accessories or consumables which are not recommended by Eppendorf.

• The device is maintained or repaired by people not authorized by Eppendorf.

• The user makes unauthorized changes to the device.

3.5 Safety instructions located on the device

Depiction Meaning Location

Hazard point

 Follow the operating manual.

The device needs to be readjusted after it has been opened.

 Do not open the device.

Rear side of the device

Bottom of the device

Installation

Eppendorf BioSpectrometer

fluorescence

English (EN)

4 Installation

4.1 Preparing installation

 Keep the transport carton and the packing material for subsequent safe transport or storage.

 Check the completeness of the delivery using the information in the delivery package (see Delivery

package on p. 11).

 Check all parts for any transport damage.

4.2 Selecting the location

Select the location for the BioSpectrometer fluorescence according to the following criteria:

• 2 grounded sockets for the BioSpectrometer fluorescence and for the printer.

• Solid laboratory bench with horizontal work surface Space requirement of the device: 50 cm (with printer: 75 cm) width, 50 cm depth.

• Temperature: 15°C to 35°C.

• Avoid temperature fluctuations (e.g, caused by open windows).

• Avoid direct sunlight.

• Humidity: 25% to 70% relative humidity.

Ensure that no objects (e.g., loose sheets, notebooks) that could impede the flow of air are positioned under the device.

4.3 Connecting the device to the mains

1. Place the BioSpectrometer fluorescence on a suitable work surface.

2. Verify that the mains/power supply voltage and mains/power frequency match the information on the name plate.

3. Connect the device to the mains/power line and switch it on with the power switch.

4. Remove the protective film from the display.

Installation

Eppendorf BioSpectrometer English (EN)

fluorescence

4.4 Connecting the printer

4.4.1 Thermal printer DPU-S445

Prerequisites

Software version 3.4.4.0 or higher is installed on the device.

Connect the thermal printer DPU-S445 to the USB port for printers.

1. Connect the printer cable with the USB port for printers 4 (see Main illustration on p. 11).

2. Connect the printer cable with the printer.

3. Connect the printer to the mains/power line using the supplied mains/power adaptor and mains/power cord (printer accessory) and switch it on.

For information on the printer, refer to the operating manual of the printer.

4.4.2 Thermal printer DPU-414

Connect the thermal printer DPU-414 to the serial printer connection.

1. Connect the printer cable to the serial printer connection 9 and tighten the locking screws.(see Main illustration on p. 11).

2. Connect the printer cable to the printer and tighten the locking screws as well.

3. Connect the printer to the mains/power line using the supplied mains/power adaptor and mains/power cord (printer accessory) and switch it on.

Information about modifying printer settings can be found in the operating manual for the printer.

The DIP switches are preset for the BioSpectrometer fluorescence according to the following table.

Tab. 4-1: Setting the DIP SW for the thermal printer

DIP SW-1 Meaning

1 (OFF) Input = Serial

2 (ON) Printing Speed = High

3 (ON) Auto Loading = ON

4 (OFF) Auto LF = OFF

5 (ON) Setting Command = Enable

6 (OFF) Printing

7 (ON) Density

8 (ON) = 100%

DIP SW-2 Meaning

1 (ON) Printing Columns = 40

2 (ON) User Font Back-up = ON

3 (ON) Character Select = Normal

DIP SW-2 Meaning

4 (ON) Zero = Normal

5 (ON) International

6 (ON) Character

7 (ON) Set

8 (OFF) = U.S.

DIP SW-3 Meaning

1 (ON) Data Length = 8 bits

2 (ON) Parity Setting = NO

3 (ON) Parity Condition = Odd

4 (OFF) Busy Control = XON/XOFF

5 (OFF) Baud

6 (ON) Rate

7 (ON) Select

8 (ON) = 9600 bps

Eppendorf BioSpectrometer

Installation

fluorescence

English (EN)

4.5 Connecting PC or USB stick for data export

You can connect a FAT 32-formatted USB stick to the USB port 4 (see Main illustration on p. 11).

Alternatively, you can connect the device for the data export directly to a PC by using a USB cable:

Prerequisites

• PC with Windows, version XP, SP2 or higher version.

• USB cable with a type A and type B plug each.

 Connect the device to the PC by using the USB cable on the USB port 8 (see Main illustration on p. 11).

• You do not need any special PC software for the data transmission: the transferred data packets are recognized by the PC like a USB stick as a removable medium. For viewing the data, you only need to open the registered data packet.

• The transmission of data to the USB stick or to the PC is started after completing the series of measurement in the print & export (see Print & export on p. 55) method step.

Installation

Eppendorf BioSpectrometer English (EN)

fluorescence

5 Operation

5.1 Overview of operating controls

Abb. 5-1: Control panel of the BioSpectrometer fluorescence

Eppendorf BioSpectrometer

Operation

fluorescence

English (EN)

Fig. 5-1: Control panel of the BioSpectrometer fluorescence

Key: Function

Keypad: Enter digits and text. Keys 1 to 9 as well as 0: When entering text, next to numbers you also can enter letters and special characters by pressing the key several times. Alternatively, you can switch to a displayed keyboard with the [Keyboard] key.

Outside of entry fields: Call up method selection.

Outside of entry fields: Call up function selection.

Softkey: Select functions. The key assignment changes along with the software dialog. The current function is displayed directly above the key on the display.

Operation

Eppendorf BioSpectrometer

fluorescence

English (EN)

Key: Function

Move the cursor to the left, right, up, down.

• Navigation between input fields.

• and keys inside an entry field: Navigate within the character string.

• and keys in a result display: Navigate between the sample results of the series of measurement.

• and keys within a graph: Navigate on the x-axis of the graph, e.g. for displaying the wavelength-dependent absorbance values in a scan.

and keys in an absorbance wavelength spectrum: Change image

section (SpectraZoom procedure) (see Tab. on p. 52).

Exit the current selection for the next higher level.

Delete entry. Within a sequence of signs, the sign on the left of the cursor is deleted

•Call up selected method or function.

• Open the selection list.

• Confirm entry or selection.

Start standard measurement.

Start blank measurement.

Start sample measurement.

Operation

Eppendorf BioSpectrometer

fluorescence

English (EN)

5.1.1 Entering text

You can enter texts when assigning method names and result units. Restriction: Only digits, letters and the underscore "_" are allowed for method names.

Entry via keyboard: Use the and cursor keys to navigate within the

entry field and to change single positions in the name. Softkeys:

• [Keyboard]: Display keyboard.

• [abc]: Change between upper and lower case letters when making entries with the keypad.

• [Save]: Save entered text.

• [Cancel]: Cancel text input.

Entry via the displayed keyboard: Use the cursor keys to select the displayed signs and respectively confirm your selection with the enter key. As for a PC key pad, you can use the "Shift" resp. the "Caps Lock" key for changing the capitalization for the next entry or for all following entries. Softkeys:

• [Numbers]: Switch to entry using the keyboard.

• [Save]: Save entered text.

• [Cancel]: Cancel text input.

Operation

Eppendorf BioSpectrometer

fluorescence

English (EN)

5.2 Inserting the cuvette

Standard rectangular glass or plastic cuvettes can be inserted in the cuvette shaft:

• External dimensions: 12.5 mm × 12.5 mm

• Height of light path: 8.5 mm higher than cuvette base

• Total height: min. 36 mm

The cuvettes must be optically transparent for the respective measuring wavelength. For measurements in the UV range, Eppendorf offers the plastic cuvette UVette which is transparent for wavelengths of 220 nm and higher and therefore also is suitable for measuring nucleic acids.

Cuvettes

Basic area 12.5 mm × 12.5 mm Min. overall height 36 mm

Min. filling level 10 mm Light path 8.5 mm Max. height of base 7 mm 0 mm Min. volume Photometry

Min. volume Fluorimetry

Eppendorf

µCuvette G1.0

See manufacturer

information

See manufacturer

information

Hellma

See manufacturer

* or similar microliter cuvette

TrayCell *

information not suitable

50 µL

60 µL

70 µL

400 µL

MacroSemi-microUltra-microUVette

Prerequisites

• The cuvette is free from contamination by dust or fingerprints and free from scratches.

• The cuvette shaft is free from particles, dust and liquid.

• The measuring volume in the cuvette is sufficient. Ensure that the minimum measuring volume has been reached.

• The measuring solution is free from particles and bubbles.

• Fluorimetry: The measuring solution is free of substances that exhibit unwanted autofluorescence or weaken the fluorescence of the substance to be examined.

• The cuvette temperature is above the temperature of the dew point that applies for the ambient conditions (humidity and temperature).

1000 µL

Operation

Eppendorf BioSpectrometer

fluorescence

English (EN)

The direction of the light path is marked with an arrow on the housing.

• Photometry: The direction of the light path from back to front is marked on the housing: "absorbance".

• Fluorimetry: The direction of the light path from right to left and back is marked on the cuvette shaft cover: "fluorescence".

Abb. 5-2: Marking of light paths

fluorescence

absorbance

height

8.5 mm

Fig. 5-2: Marking of light paths

1. Position the cuvette so that the optical window of the cuvette is pointing towards the direction of the light path.

2. When inserting the cuvette, press it completely to the bottom against the slight resistance.

3. Fluorimetry: Close the cuvette shaft cover prior to measurement.

5.3 Summary of the measuring procedure

5.3.1 Preparing the measurement

1. Switch on the device and, if required, the printer.

The device performs a self test (taking approx. 1 minute) and displays the method selection.

2. Make ready the cuvettes for the measurements (see Inserting the cuvette on p. 24).

3. Prepare the measuring solutions for measuring the blank values, if required, also the standards and the samples.

4. Open the cover of the cuvette shaft.

You should not use any measuring solution for standards and samples with a lower absorbance than 0.02 to 0.03 A (e.g. dsDNA concentration between 1.0 and 1.5 μg/mL). The detection limit of the device may be significantly lower, nevertheless, the impact of disturbances from the measuring solutions (particles, bubbles, turbidity) on the reliability of the result is very high for these low absorbance values.

Operation

Eppendorf BioSpectrometer English (EN)

fluorescence

5.3.2 Measuring procedure

5.3.2.1 Selecting a method

 Use the cursor keys to select the desired

method and call up the method with the enter

key. For an overview and a detailed description of the methods, refer to the next chapter (see Methods on p. 31).

Wizard: The wizard at the top of the display will take you through the method procedure step-by-step. Help box: You will receive help texts in the lower right of the display during each step of the procedure. Softkeys: The [< Back] and [Next >] softkeys allow you to move between method steps in the wizard.

5.3.2.2 Checking parameters

 Check the parameter setting. The [Page dn] and

[Page up] softkeys allow you to call up the

parameter list pages. You can modify and save

parameters using [Edit].

5.3.2.3 Measuring the blank and standards

For evaluations without standards (e.g. DNA measurements), this method step is omitted.

Operation

Eppendorf BioSpectrometer

1. Start by measuring a blank (blank key).

2. Then measure all standards one by one

(standard key). The display always marks the standard that is to be measured next. Use the [Graph] resp. [Table] softkey to change the result view.

fluorescence

English (EN)

5.3.2.4 Measuring samples

 Press [Next] to accept the evaluation calculated

from the standard results.

 The sample key is used for measuring your

samples consecutively. Blank results will remain saved for the duration of one series of measurement. However, a new blank measurement always is possible. (The adjacent figure shows a measuring procedure with evaluation via the standard curve and, in addition to the sample result, the graph of the standard evaluation.)

Operation

Eppendorf BioSpectrometer English (EN)

fluorescence

5.3.2.5 Finalizing the method

5.3.2.6 Optional: process results

1. Press [Finish], to complete the measuring series

and return to the method selection.

2. After all measurements have been completed,

switch off the device and close the cuvette shaft

cover to protect the cuvette shaft from

contamination.

5.3.2.7 Printing and exporting

For some methods, you can postprocess the results in the process results method step. For example, you can use the SpectraZoom zoom function in the spectra.

 Use the and cursor keys for selecting

systematically any results of the series of

measurement for postprocessing.

1. Compose data packets for all samples or for

selected samples.

2. Print the data, save them to a USB stick or

transfer them to a PC via a USB cable.

5.3.3 Important measurement instructions

Check for each measurement:

• For plastic cuvettes: How many consecutive measurements can be reliably carried out in the cuvette?

• Measure the cuvette blank value before the sample or standard measurements in order to compensate the cuvette blank value in addition to the reagent blank.

• Blank results remain saved for one measuring series, but a new blank result measurement can be performed at any time, even between sample measurements.

• The displayed absorbance and RFU values always correspond to the directly measured values. The dilution or cuvette factor as well as background absorbances only will be incorporated for the following result calculation (see Absorbance values on p. 87).

• The measuring result is typically displayed 2 to 3 seconds after a measurement has been started. If a small amount of light reaches the receiver (high absorbance values or low RFU values), the measuring time can be automatically extended by up to 9 seconds (photometry) or 6 seconds (fluorimetry) in order to increase the precision of the measurement.

• Observe that the measured absorbance values do not exceed the upper limit of the photometric measuring range. In this case, reject the measuring result. The upper limit of the photometric measuring range does not only depend on the wavelength (see Photometric properties on p. 84) but also on the cuvette blank. Ultra-micro cuvettes with a small diaphragm, such as TrayCell (Hellma), may have a cuvette blank of approx. A = 1. The available photometric measuring range is reduced by this amount. You can estimate the cuvette blank by measuring the cuvette filled with demineralized water as a sample in comparison with the empty cuvette shaft as a blank. The cuvette blank of the Eppendorf μCuvette G1.0 is negligible (approximately A = 0). Fluorimetry: An increased autofluorescence of the cuvette (typical for plastic cuvettes) may restrict the available measuring range.

• After the measurement, remove the measuring solution completely before filling in the next measuring solution in order to minimize carry-over. If a carry-over from one sample to the next sample can be expected due to a high concentration difference, rinse the cuvette between the measurements.

• If the temperature between the lamp and the ambience differs, photometric drift may occur. Therefore a device from a colder ambience first has to be adjusted to the ambient temperature. Avoid quick changes of temperature. Carry out a new blank measurement for a long series of measurements or measurements over a long period of time.

Eppendorf BioSpectrometer

Operation

fluorescence

English (EN)

Operation

Eppendorf BioSpectrometer English (EN)

fluorescence

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