Thermo Fisher Scientific resDNASEQ User Manual

resDNASEQ™ Quantitative DNA Kits

USER GUIDE

for use with: resDNASEQ™ Quantitative CHO DNA Kit resDNASEQ™ Quantitative E. coli DNA Kit resDNASEQ™ Quantitative HEK293 DNA Kit resDNASEQ™ Quantitative Human DNA Kit resDNASEQ™ Quantitative MDCK DNA Kit resDNASEQ™ Quantitative NS0 DNA Kit resDNASEQ™ Quantitative Pichia DNA Kit resDNASEQ™ Quantitative Sf9 and Baculovirus DNA Kit resDNASEQ™ Quantitative Vero DNA Kit

Catalog Numbers 4402085, 4458435, A46014, A26366, 4464335, 4458441, 4464336, A46066, A41797

Publication Number 4469836

Revision G

For Research Use Only. Not for use in diagnostic procedures.

Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. 4469836

Revision Date Description

F 17 December 2019 Update to include the resDNASEQ™ Quantitative HEK293 DNA Kit

5 August 2020 Update to include the resDNASEQ™ Quantitative Sf9 and Baculovirus

DNA Kit (Cat. No. A46066).

(Cat. No. A46014).

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed.

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Product information ................................................................... 5

Product description ............................................................. 5

Contents ....................................................................... 6

Required materials not supplied ................................................. 11

Workﬂow ..................................................................... 12

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Methods .............................................................................. 13

Prepare the control DNA serial dilutions for the standard curve ...................... 13

Guidelines for standard dilutions ............................................. 13

Prepare the control DNA serial dilutions ...................................... 13

Prepare the PCR reaction mix ................................................... 15

Prepare the PCR plate .......................................................... 16

Setup, run, and analyze samples with AccuSEQ™ Software on the QuantStudio

5 Real‑Time PCR Instrument .................................................. 18

Create a resDNASEQ™ experiment ........................................... 18

Start the run .............................................................. 22

Analyze the results ......................................................... 23

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APPENDIX A Troubleshooting .................................................... 24

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APPENDIX B Use the kit with the 7500 Fast Real‑Time PCR

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APPENDIX C Use the kit with 7500 System SDS Software v1.5.1 ............ 31

resDNASEQ

Instrument and AccuSEQ™ software v2.x ....................................... 26

Required materials not supplied ................................................. 26

Create a plate document in the AccuSEQ™ software ................................ 27

Run the plate .................................................................. 29

Analyze the results ............................................................. 29

Create the plate document, run the plate, and analyze the results with 7500 Fast

SDS software ................................................................ 31

Create a plate document ................................................... 31

Run the plate .............................................................. 34

Analyze the results ......................................................... 35

™

Quantitative DNA Kits User Guide

Contents

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APPENDIX D Good laboratory practices ....................................... 36

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APPENDIX E Safety ............................................................... 37

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Documentation and support ....................................................... 40

References

Work area setup and lab design .................................................. 36

Good laboratory practices for PCR and RT-PCR ................................... 36

Avoiding false positives due to cross-contamination ........................... 36

Chemical safety ................................................................ 38

Biological hazard safety ......................................................... 39

Related documentation ......................................................... 40

Customer and technical support ................................................. 41

Limited product warranty ........................................................ 41

resDNASEQ™ Quantitative DNA Kits User Guide

IMPORTANT! Before using this product, read and understand the information in the

“Safety” appendix in this document.

Product description

The resDNASEQ™ Quantitative DNA Kits are used to quantitate residual DNA from CHO, E. coli, HEK293, Human, MDCK, NS0, Pichia, Sf9 and Baculovirus, and Vero in cell lines which are used for production of biopharmaceutical products. Use the kit after you extract host‑cell DNA from test samples. For extraction information, see the PrepSEQ™ Residual DNA Sample Preparation Kit User Guide (Pub. No. 4469838).

The resDNASEQ™ Quantitative DNA Kits use TaqMan™ quantitative PCR to perform rapid, speciﬁc quantitation of sub-picogram levels of residual host‑cell DNA. The assay is accurate and reliable across a broad range of sample types, from in-process samples to ﬁnal product.

Product information

To generate the standard curve used to quantitate the DNA in test samples, the CHO, MDCK, NS0, and Vero assays require six dilutions (from 30 fg to 3 ng per reaction) and the E. coli, HEK293, Human, Pichia, and Sf9 and Baculovirus assays require ﬁve dilutions (300 fg to 3 ng per reaction). Control DNA for standard curve generation is included in the kits. In addition, the kits use an internal positive control (IPC) to evaluate the performance of each PCR reaction.

resDNASEQ

™

Quantitative DNA Kits User Guide

Product information

Contents

Table 1 resDNASEQ™ Quantitative CHO DNA Kit (Cat. No. 4402085)

Contents Amount Storage

resDNASEQ™ CHO DNA Control

CHO DNA Control, 30 ng/µL 40 µL –25°C to –15°C

DNA Dilution Buer (DDB)

7 mL

resDNASEQ™ CHO Real-Time PCR Reagents

2X Environmental Master Mix

2 × 0.75 mL

10X CHO DNA Real-Time PCR Assay Mix 300 µL −25°C to −15°C, protect from light

Negative Control (water)

1.0 mL

–25°C to –15°C

2−8°C after ﬁrst use

–25°C to −15°C before ﬁrst use, protect from light

2−8°C after ﬁrst use, protect from light

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

Table 2 resDNASEQ™ Quantitative E. coli DNA Kit (Cat. No. 4458435)

Contents

Box 1, resDNASEQ™ Real-Time PCR Reagents

2X Environmental Master Mix

Amount Storage

–25°C to –15°C before ﬁrst use, protect from light

2 × 0.75 mL

2−8°C after ﬁrst use, protect from light

Negative Control (water)

1.0 mL

10X E. coli DNA Assay Mix 300 µL –25°C to –15°C, protect from light

Box 2, resDNASEQ™ E. coli DNA Control

E. coli DNA Control, 30 ng/µL 40 µL –25°C to –15°C

DNA Dilution Buer (DDB)

7 mL

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

resDNASEQ™ Quantitative DNA Kits User Guide

Product information

Table 3 resDNASEQ™ Quantitative HEK293 DNA Kit (Cat. No. A46014)

Contents Amount Storage

resDNASEQ™ HEK293 DNA Control

HEK293 DNA Control, 30 ng/µL 40 µL –25°C to –15°C

Contents

DNA Dilution Buer (DDB)

7 mL

resDNASEQ™ HEK293 DNA Real-Time PCR Reagents

2X Environmental Master Mix

2 × 0.75 mL

10X HEK293 Assay Mix 300 µL –25°C to –15°C, protect from light

Negative Control (water)

1.0 mL

–25 to –15°C before ﬁrst use

2−8°C after ﬁrst use

–25°C to –15°C before ﬁrst use, protect from light

2−8°C after ﬁrst use, protect from light

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

Table 4 resDNASEQ™ Quantitative Human DNA Kit (Cat. No. A26366)

Contents

resDNASEQ™ Human DNA Control

Human DNA Control, 30 ng/µL 40 µL –25°C to –15°C

DNA Dilution Buer (DDB)

Amount Storage

–25 to –15°C before ﬁrst use

7 mL

2−8°C after ﬁrst use

resDNASEQ™ Human Real-Time PCR Reagents

2X Environmental Master Mix

2 × 0.75 mL

10X Human DNA Assay Mix 300 µL –25°C to –15°C, protect from light

Negative Control (water)

1.0 mL

resDNASEQ™ Quantitative DNA Kits User Guide

–25°C to –15°C before ﬁrst use, protect from light

2−8°C after ﬁrst use, protect from light

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

Product information

Contents

Table 5 resDNASEQ™ Quantitative MDCK DNA Kit (Cat. No. 4464335)

Contents Amount Storage

Box 1, resDNASEQ™ Real-Time PCR Reagents

2X Environmental Master Mix

2 × 0.75 mL

Negative Control (water)

1.0 mL

10X MDCK DNA Assay Mix 300 µL –25°C to –15°C, protect from light

Box 2, resDNASEQ™ MDCK DNA Control

MDCK DNA Control, 30 ng/µL 40 µL –25°C to –15°C

DNA Dilution Buer (DDB)

7 mL

–25°C to –15°C before ﬁrst use, protect from light

2−8°C after ﬁrst use, protect from light

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

Table 6 resDNASEQ™ Quantitative NS0 DNA Kit (Cat. No. 4458441)

Contents

Box 1, resDNASEQ™ Real-Time PCR Reagents

2X Environmental Master Mix

Amount Storage

–25°C to –15°C before ﬁrst use, protect from light

2 × 0.75 mL

2−8°C after ﬁrst use, protect from light

Negative Control (water)

1.0 mL

10X NS0 DNA Assay Mix 300 µL –25°C to –15°C, protect from light

Box 2, resDNASEQ™ NS0 DNA Control

NS0 DNA Control, 30 ng/µL 40 µL –25°C to –15°C

DNA Dilution Buer (DDB)

7 mL

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

resDNASEQ™ Quantitative DNA Kits User Guide

Table 7 resDNASEQ™ Quantitative Pichia DNA Kit (Cat. No. 4464336)

Contents Amount Storage

Box 1, resDNASEQ™ Real-Time PCR Reagents

Product information

Contents

2X Environmental Master Mix

2 × 0.75 mL

Negative Control (water)

1.0 mL

10X Pichia DNA Assay Mix 300 µL –25°C to –15°C, protect from light

Box 2, resDNASEQ™ Pichia DNA Control

Pichia DNA Control, 30 ng/µL 40 µL –25°C to –15°C

DNA Dilution Buer (DDB)

7 mL

–25°C to –15°C before ﬁrst use, protect from light

2−8°C after ﬁrst use, protect from light

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

Table 8 resDNASEQ™ Quantitative Sf9 and Baculovirus DNA Kit (Cat. No. A46066)

Contents

Box 1, resDNASEQ™ Real-Time PCR Reagents

2X Environmental Master Mix

Amount Storage

–25°C to –15°C before ﬁrst use, protect from light

2 × 0.75 mL

2−8°C after ﬁrst use, protect from light

Negative Control (water)

10X Sf9 + Baculovirus DNA Assay Mix 300 µL –25°C to –15°C, protect from light

Box 2, resDNASEQ™ Sf9 and Baculovirus DNA Control

Multiplex DNA Control with:

• Sf9 DNA Control, 30 ng/µL

• Baculovirus DNA Control, 30 ng/µL

DNA Dilution Buer (DDB)

resDNASEQ™ Quantitative DNA Kits User Guide

1.0 mL

40 µL –25°C to –15°C

7 mL

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

–25°C to –15°C before ﬁrst use

2−8°C after ﬁrst use

Product information

Contents

Table 9 resDNASEQ™ Quantitative Vero DNA Kit (Cat. No. A41797)

Contents Amount Storage

Box 1, resDNASEQ™ Real-Time PCR Reagents

2X Environmental Master Mix

–25°C to –15°C before ﬁrst use, protect from light

2 × 0.75 mL

2−8°C after ﬁrst use, protect from light

Negative Control (water)

–25°C to –15°C before ﬁrst use

1.0 mL 2−8°C after ﬁrst use

10X Vero DNA Assay Mix 300 µL –25°C to –15°C, protect from light

Box 2, resDNASEQ™ Vero DNA Control

Vero DNA Control, 30 ng/µL 40 µL –25°C to –15°C

DNA Dilution Buer (DDB)

–25°C to –15°C before ﬁrst use

7 mL

2−8°C after ﬁrst use

resDNASEQ™ Quantitative DNA Kits User Guide

Required materials not supplied

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier.

Item Source

Instrument

QuantStudio™ 5 Real‑Time PCR System with AccuSEQ Real‑Time PCR Software v3.1 or later

Consumables

Product information

Required materials not supplied

™

Contact your local sales representative.

MicroAmp™ Optical 96-Well Reaction Plate with Barcode, 20 plates, 0.1‑mL wells; for use with Applied Biosystems™ 7500 Fast Real-Time PCR System

MicroAmp™ 96-Well Base N8010531, 10 bases

MicroAmp™ Optical Adhesive Film 4311971, 100 covers

MicroAmp™ Adhesive Film Applicator 4333183, 5 applicators

Miscellaneous items

Disposable gloves Major lab supplier (MLS)

Pipettes MLS

Aerosol-resistant micropipette tips MLS

For the PCR plate: Fisher Scientiﬁc™ Mini Plate Spinner Centrifuge, 120- or 230‑volt

Nonstick, RNaseZap™-free Microfuge Tubes, 1.5‑mL (1 box; 250 tubes/box)

14-100-143 (120‑volt), 14-100-141 (230‑volt)

4346906

4360954, 25 covers

AM12450

resDNASEQ™ Quantitative DNA Kits User Guide

Product information

Workﬂow

Prepare the control DNA serial dilutions

(page 13)

▼

Prepare the PCR reaction mix

(page 15)

▼

Prepare the PCR plate

(page 16)

▼

AccuSEQ™ Software v3.0 for the QuantStudio™ 5 Instrument

Create a resDNASEQ™ experiment

(page 18)

▼

Start the run

(page 22)

▼

Analyze the results

(page 23)

resDNASEQ™ Quantitative DNA Kits User Guide

Methods

Prepare the control DNA serial dilutions for the standard curve

Guidelines for standard dilutions

•

Prepare the standard curve and the test samples in dierent areas of the lab.

•

Use dierent sets of pipettors for test sample preparation and for standard curve preparation and aliquoting to avoid cross-contamination of test samples.

•

Vortex each tube to mix the contents thoroughly before each dilution step.

•

Brieﬂy centrifuge to collect all the liquid at the bottom before making the next dilution.

Prepare the control DNA serial dilutions

Label nonstick 1.5‑mL microfuge tubes: NTC, SD1, SD2, SD3, SD4, SD5.

For CHO, Vero, MDCK, and NS0 kits, label an additional tube with SD6.

Add 50 µL of DNA Dilution Buer (DDB) to tube NTC. Put aside.

Add 990 µL of DNA Dilution Buer (DDB) to tube SD1.

Add 450 µL of DNA Dilution Buer (DDB) to tubes SD2, SD3, SD4, SD5, and (for CHO, Vero, MDCK, and NS0 only) SD6.

Remove the tube of DNA control (30 ng/µL) from the freezer.

After the DNA thaws, vortex it gently for 2 seconds, then brieﬂy centrifuge.

Perform the serial dilutions:

Add 10 µL of the DNA control to the tube that is labeled SD1, then vortex thoroughly and brieﬂy centrifuge.

Transfer 50 µL of the DNA from tube SD1 to tube SD2, then vortex thoroughly and brieﬂy centrifuge.

resDNASEQ

™

Quantitative DNA Kits User Guide

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