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
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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 |
G | 5 August 2020 | Update to include the resDNASEQ™ Quantitative Sf9 and Baculovirus |
DNA Kit (Cat. No. A46066). | ||
F | 17 December 2019 | Update to include the resDNASEQ™ Quantitative HEK293 DNA Kit |
(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. ©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
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Product information ................................................................... | 5 |
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
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 |
APPENDIX B Use the kit with the 7500 Fast Real Time PCR | |
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 |
■ APPENDIX C Use the kit with 7500 System SDS Software v1.5.1 ............ | 31 |
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
resDNASEQ™ Quantitative DNA Kits User Guide | 3 |
Contents | |
■ APPENDIX D Good laboratory practices ....................................... | 36 |
Work area setup and lab design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Avoiding false positives due to cross-contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
■ APPENDIX E Safety ............................................................... | 37 |
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . | 38 |
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . | 39 |
Documentation and support ....................................................... | 40 |
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
References
4 | resDNASEQ™ Quantitative DNA Kits User Guide |
Product information
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.
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 | 5 |
Product information
Contents
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 Buffer (DDB) | 7 mL | –25°C to –15°C |
2−8°C after first use | ||
resDNASEQ™ CHO Real-Time PCR Reagents | ||
2X Environmental Master Mix | –25°C to −15°C before first use, | |
2 × 0.75 mL | protect from light | |
2−8°C after first use, protect from | ||
light | ||
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 before first use |
2−8°C after first use | ||
Table 2 resDNASEQ™ Quantitative E. coli DNA Kit (Cat. No. 4458435) | ||
Contents | Amount | Storage |
Box 1, resDNASEQ™ Real-Time PCR Reagents | ||
2X Environmental Master Mix | –25°C to –15°C before first use, | |
2 × 0.75 mL | protect from light | |
2−8°C after first use, protect from | ||
light | ||
Negative Control (water) | 1.0 mL | –25°C to –15°C before first use |
2−8°C after first use | ||
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 Buffer (DDB) | 7 mL | –25°C to –15°C before first use |
2−8°C after first use | ||
6 | resDNASEQ™ Quantitative DNA Kits User Guide |
Product information
Contents
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 | |
DNA Dilution Buffer (DDB) | 7 mL | –25 to –15°C before first use | |
2−8°C after first use | |||
resDNASEQ™ HEK293 DNA Real-Time PCR Reagents | |||
2X Environmental Master Mix | –25°C to –15°C before first use, | ||
2 × 0.75 mL | protect from light | ||
2−8°C after first use, protect from | |||
light | |||
10X HEK293 Assay Mix | 300 µL | –25°C to –15°C, protect from light | |
Negative Control (water) | 1.0 mL | –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 | Amount | Storage | |
resDNASEQ™ Human DNA Control | |||
Human DNA Control, 30 ng/µL | 40 µL | –25°C to –15°C | |
DNA Dilution Buffer (DDB) | 7 mL | –25 to –15°C before first use | |
2−8°C after first use | |||
resDNASEQ™ Human Real-Time PCR Reagents | |||
2X Environmental Master Mix | –25°C to –15°C before first use, | ||
2 × 0.75 mL | protect from light | ||
2−8°C after first use, protect from | |||
light | |||
10X Human DNA Assay Mix | 300 µL | –25°C to –15°C, protect from light | |
Negative Control (water) | 1.0 mL | –25°C to –15°C before first use | |
2−8°C after first use | |||
resDNASEQ™ Quantitative DNA Kits User Guide | 7 |
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 | –25°C to –15°C before first use, | |
2 × 0.75 mL | protect from light | |
2−8°C after first use, protect from | ||
light | ||
Negative Control (water) | 1.0 mL | –25°C to –15°C before first use |
2−8°C after first use | ||
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 Buffer (DDB) | 7 mL | –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 | Amount | Storage |
Box 1, resDNASEQ™ Real-Time PCR Reagents | ||
2X Environmental Master Mix | –25°C to –15°C before first use, | |
2 × 0.75 mL | protect from light | |
2−8°C after first use, protect from | ||
light | ||
Negative Control (water) | 1.0 mL | –25°C to –15°C before first use |
2−8°C after first use | ||
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 Buffer (DDB) | 7 mL | –25°C to –15°C before first use |
2−8°C after first use | ||
8 | resDNASEQ™ Quantitative DNA Kits User Guide |
Product information
Contents
Table 7 resDNASEQ™ Quantitative Pichia DNA Kit (Cat. No. 4464336)
Contents | Amount | Storage | |
Box 1, resDNASEQ™ Real-Time PCR Reagents | |||
2X Environmental Master Mix | –25°C to –15°C before first use, | ||
2 × 0.75 mL | protect from light | ||
2−8°C after first use, protect from | |||
light | |||
Negative Control (water) | 1.0 mL | –25°C to –15°C before first use | |
2−8°C after first use | |||
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 Buffer (DDB) | 7 mL | –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 | Amount | Storage | |
Box 1, resDNASEQ™ Real-Time PCR Reagents | |||
2X Environmental Master Mix | –25°C to –15°C before first use, | ||
2 × 0.75 mL | protect from light | ||
2−8°C after first use, protect from | |||
light | |||
Negative Control (water) | 1.0 mL | –25°C to –15°C before first use | |
2−8°C after first use | |||
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 | 40 µL | –25°C to –15°C | |
• Baculovirus DNA Control, 30 ng/µL | |||
DNA Dilution Buffer (DDB) | 7 mL | –25°C to –15°C before first use | |
2−8°C after first use | |||
resDNASEQ™ Quantitative DNA Kits User Guide | 9 |
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, | |
2 × 0.75 mL | protect from light | |
2−8°C after first use, protect from | ||
light | ||
Negative Control (water) | 1.0 mL | –25°C to –15°C before first use |
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 Buffer (DDB) | 7 mL | –25°C to –15°C before first use |
2−8°C after first use | ||
10 | resDNASEQ™ Quantitative DNA Kits User Guide |
Product information
Required materials not supplied
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™ | Contact your local sales representative. |
Real Time PCR Software v3.1 or later | |
Consumables | |
MicroAmp™ Optical 96-Well Reaction Plate with | 4346906 |
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 |
4360954, 25 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 | 14-100-143 (120 volt), 14-100-141 (230 volt) |
Centrifuge, 120or 230 volt | |
Nonstick, RNaseZap™-free Microfuge Tubes, 1.5 mL | AM12450 |
(1 box; 250 tubes/box) | |
resDNASEQ™ Quantitative DNA Kits User Guide | 11 |
Product information
Workflow
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)
12 | 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 different areas of the lab.
•Use different 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
1.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.
2.Add 50 µL of DNA Dilution Buffer (DDB) to tube NTC. Put aside.
3.Add 990 µL of DNA Dilution Buffer (DDB) to tube SD1.
4.Add 450 µL of DNA Dilution Buffer (DDB) to tubes SD2, SD3, SD4, SD5, and (for CHO, Vero, MDCK, and NS0 only) SD6.
5.Remove the tube of DNA control (30 ng/µL) from the freezer.
6.After the DNA thaws, vortex it gently for 2 seconds, then briefly centrifuge.
7.Perform the serial dilutions:
a.Add 10 µL of the DNA control to the tube that is labeled SD1, then vortex thoroughly and briefly centrifuge.
b.Transfer 50 µL of the DNA from tube SD1 to tube SD2, then vortex thoroughly and briefly centrifuge.
resDNASEQ™ Quantitative DNA Kits User Guide | 13 |
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