Thermo Fisher Scientific PrepSEQ User Manual

PrepSEQ™ Residual DNA Sample Preparation
Kit

USER GUIDE

Automated and manual protocols for preparation of samples for
use with resDNASEQ™ Quantitative DNA Kits

Catalog Number 4413686

Publication Number 4469838

Revision F

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. 4469838

Revision Date Description

F

E 12 Dec 2019 Added the resDNASEQ™ Quantitative HEK293 DNA Kit

5 August 2020

Added 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.

©2020 Thermo Fisher Scientific Inc. All rights reserved.

Contents

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CHAPTER 1 Product information .................................................. 5

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

Contents and storage ............................................................ 5

Required materials not supplied for manual protocols ................................ 6

Required materials not supplied for automated protocols ............................. 7

Workflow ....................................................................... 9

■

CHAPTER 2 Prepare the reagents and samples ................................ 10

Prepare the reagents: before first use of the kit .................................... 10

Magnetic beads ........................................................... 10

Binding Solution ........................................................... 10

Wash Buer Concentrate ................................................... 10

Prepare reagents: before each use of the kit ....................................... 10

Proteinase K (PK) mix ...................................................... 10

Lysis solution ............................................................. 11

Guidelines for optimal DNA yields ................................................ 11

Sample preparation guidelines ................................................... 12

Sample dilution (if necessary) ............................................... 12

Triplicate extractions ....................................................... 12

Extraction control guidelines ................................................ 13

■

■

PrepSEQ

CHAPTER 3 Manual protocol for residual DNA extraction ..................... 14

Digest the test samples and controls ............................................. 14

Bind the DNA .................................................................. 14

Wash the DNA ................................................................. 15

Elute the DNA ................................................................. 16

CHAPTER 4 Automated protocol for residual DNA extraction ................ 17

Before each use of the kit ....................................................... 17

Ensure that you have the correct plates ....................................... 17

Prepare the plates ......................................................... 17

Prepare the lysis plate .......................................................... 18

Process samples on the instrument .............................................. 18

™

Residual DNA Sample Preparation Kit User Guide

3

Contents

■

■

■

■

APPENDIX A Troubleshooting .................................................... 20

APPENDIX B Good laboratory practices ....................................... 21

Good laboratory practices for PCR and RT-PCR ................................... 21

Avoiding false positives due to cross-contamination ........................... 21

Plate layout suggestions ....................................................... 21

APPENDIX C Safety ............................................................... 22

Chemical safety ................................................................ 23

Biological hazard safety ......................................................... 24

Documentation and support ....................................................... 25

Related documentation ......................................................... 25

Customer and technical support ................................................. 25

Limited product warranty ........................................................ 26

4

PrepSEQ™ Residual DNA Sample Preparation Kit User Guide

1

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

“Safety” appendix in this document.

Product description

The PrepSEQ™ Residual DNA Sample Preparation Kit extracts residual DNA from
products that are produced in cell lines such as CHO, E. coli, HEK293, Human,
MDCK, NS0, Pichia, Sf9 and Baculovirus, and Vero. The kit uses chemical lysis
and magnetic beads to extract genomic DNA from diverse sample types, including
samples that contain high protein and low DNA concentration.

For quantification of host-cell line residual DNA, we recommend using the
resDNASEQ™ Quantitative DNA Kits as described in the resDNASEQ™ Quantitative
DNA Kits User Guide (Pub. No. 4469836). To ensure accurate quantitative results,
extract each sample in triplicate and perform a single PCR reaction for each
extraction.

Product information

Contents and storage

The kit contains reagents sucient for 100 extractions.

Table 1 PrepSEQ™ Residual DNA Sample Preparation Kit (Cat. No. 4413686)

Contents

Box 1, PrepSEQ™ Nucleic Acid Extraction Kit

Lysis Buer 2 × 50 mL

Binding Solution (Isopropanol), empty bottle 1

Wash Buer Concentrate 2 × 26 mL

Elution Buer 25 mL

Proteinase K (PK) Buer

Can be used for existing validated manual
protocols.

Amount Storage

50 mL

Room temperature

PrepSEQ™ Residual DNA Sample Preparation Kit User Guide

5

Chapter 1 Product information

1

Required materials not supplied for manual protocols

Table 1 PrepSEQ Residual DNA Sample Preparation Kit (Cat. No. 4413686) (continued)

Contents Amount Storage

Proteinase K (PK) Buer II

Recommended for new manual protocols.

Required for automated protocols.

Box 2, PrepSEQ™ Nucleic Acid Extraction Kit

Magnetic Particles 2 × 1.5 mL Room temperature

Box 3, PrepSEQ™ Nucleic Acid Extraction Kit

Proteinase K, 20 mg/mL 1.25 mL –20°C or below

PrepSEQ™ Residual DNA Sample Preparation Kit

Proteinase K, 20 mg/mL 1.25 mL

Yeast tRNA, 10 mg/mL 0.5 mL

Glycogen, 5 mg/mL 2 × 1.0 mL

[1]

Also sold separately (Cat. No. 4415320).

[1]

Room temperature50 mL

–20°C or below

Required materials not supplied for manual protocols

Unless otherwise indicated, all materials are available through thermofisher.com.
MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Item

Equipment

Pharma Magnetic Stand-96 or

Magnetic stand, 16-position

Block heater for use with 2‑mL tubes.

Manual DNA extraction involves two incubations at
dierent settings, so two heaters may be convenient.

Benchtop microcentrifuge for 1.5‑mL and 2‑mL tubes MLS

Vortex-Genie™ 2T Mixer VWR™ 14216-188, VWR™ 14216-186

Vortex Adapter-60, for use with the Vortex-Genie

Consumables

Disposable gloves MLS

Aerosol-resistant micropipette tips MLS

™

Major laboratory supplier (MLS)

Source

A31543

12321D

AM10014

6

PrepSEQ™ Residual DNA Sample Preparation Kit User Guide

Chapter 1

Required materials not supplied for automated protocols

Item Source

Product information

1

PIPETMAN™ Pipettors, P1000, P200, P20 and P10:

•

Positive-displacement

•

Air-displacement

•

Multichannel

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

Safe-Lock PCR clean microcentrifuge tubes, round­bottom, 2‑mL

Reagents

Ethanol, 95%

MLS

AM12450

VWR™ 62111-754

MLS

IMPORTANT! Do not use denatured ethanol. It

contains components that are not compatible with the
protocol.

Isopropanol, 100% MLS

5 M NaCl and 1 N NaOH solutions MLS

Hydrochloric acid (HCI) MLS

PBS solution, 1X, pH 7.4, free of Mg and Ca (if needed to
dilute samples before DNA extraction)

14190094

Required materials not supplied for automated protocols

Unless otherwise indicated, all materials are available through thermofisher.com.
MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Table 2 Pharma KingFisher™ Flex-96 Deep Well Magnetic Particle Processor
(Cat. No. A31508) and Pharma MagMAX™ Express-96 instrument

Item

Pharma MagMAX™ Express-96 DW plate A31540

Pharma MagMAX™ Express-96 Deep-Well Tip
Combs

Pharma KingFisher™ Flex Magnetic Head for 96
Deep-Well Plate

Pharma MagMAX™ 96 PCR Well Magnetic Head 4472991

Pharma MagMAX™ Express-96 Standard Plates A31541

[1]

This instrument is no longer available for purchase

Source

A31537

A31542

[1]

accessories

PrepSEQ™ Residual DNA Sample Preparation Kit User Guide

7

Chapter 1 Product information

1

Required materials not supplied for automated protocols

Table 3 Additional materials

Consumables

Disposable gloves MLS

Aerosol-resistant micropipette tips MLS

Item Source

PIPETMAN™ Pipettors, P1000, P200, P20 and P10:

•

Positive-displacement

•

Air-displacement

•

Multichannel

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

Reagents

Ethanol, 95%

MLS

AM12450

MLS

IMPORTANT! Do not use denatured ethanol. It

contains components that are not compatible with
the protocol.

Isopropanol, 100% MLS

5 M NaCl and 1 N NaOH solutions MLS

Hydrochloric acid (HCI) MLS

PBS solution, 1X, pH 7.4, free of Mg and Ca (if
needed to dilute samples before DNA extraction)

14190094

8

PrepSEQ™ Residual DNA Sample Preparation Kit User Guide

Workflow

Chapter 1 Product information

Workflow

1

Manual residual DNA extraction

Digest the test samples and controls

(page 14)

▼ ▼

Bind the DNA (page 14) Prepare the lysis plate (page 18)

▼ ▼

Wash the DNA (page 15)

▼

Elute the DNA (page 16)

Automated residual DNA extraction

Prepare the plates (page 17)

Process samples on the instrument

(page 18)

PrepSEQ™ Residual DNA Sample Preparation Kit User Guide

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