Thermo Fisher Scientific ViralSEQ Lentivirus Titer Kits User guide

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

ViralSEQ™ Lentivirus Titer Kits

USER GUIDE

for use with:

ViralSEQ™ Lentivirus Proviral DNA Titer Kit

ViralSEQ™ Lentivirus Physical Titer Kit

Catalog NumbersA53561, A53562, A52597, and A52598

Publication Number MAN0026126

Revision B.0

Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United Kingdom

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.MAN0026126

Revision Date Description

B.0 2 September 2022 The ViralSEQ™ Lentivirus Physical Titer Kit was added to the guide,

including:

•Updates to the product description. kit contents and storage, and

workﬂows

•“Serial dilutions and sample preparation for the ViralSEQ™

Lentivirus Physical Titer Kit” on page19

•Chapter 4, “Set up, run, and review ViralSEQ™ Lentivirus Physical

Titer Kit experiments”

•AppendixB, “Use the ViralSEQ™ Lentivirus Physical Titer Kit with

the 7500 Fast Real‑Time PCR Instrument and AccuSEQ™ software

v2.x”

A.0 17 July 2022 New document for the ViralSEQ™ Lentivirus Proviral DNA Titer Kit

(Cat.No.A53561).

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. Vortex-

Genie is a trademark of Scientiﬁc Industries. Windows and Excel are trademarks of Microsoft Corporation. TaqMan is a registered

trademark of Roche Molecular Systems, Inc., used under permission and license.

Contents

■CHAPTER1Productinformation .................................................. 7

Productdescription ............................................................. 7

Contents and storage ............................................................ 8

Required materials notsupplied ................................................. 10

Lentivirus production ........................................................... 13

Kit controls .................................................................... 13

Workﬂow: ViralSEQ™ Lentivirus Physical TiterKit ................................... 15

Workﬂow: ViralSEQ™ Lentivirus Proviral DNA TiterKit ............................... 16

■CHAPTER2Prepare reagents and serialdilutions ............................. 17

Prepare the PrepSEQ™ reagents: before ﬁrst use of thekit .......................... 17

Magneticbeads ........................................................... 17

BindingSolution ........................................................... 17

Wash Buer Concentrate ................................................... 17

Prepare PrepSEQ™ reagents: before each use of thekit ............................. 18

Proteinase K (PK)mix ...................................................... 18

Lysissolution ............................................................. 18

Guidelines for optimalyields ..................................................... 19

Serial dilutions and sample preparation for the ViralSEQ™ Lentivirus Physical TiterKit .. 19

Prepare the Lentivirus RNA control serialdilutions ............................. 19

Extraction controlguidelines ................................................ 20

Prepare the extraction/ recovery control (ERC) for the ViralSEQ™ Lentivirus

Physical TiterKit ......................................................... 21

Triplicate extractions ....................................................... 22

Serial dilutions and sample preparation for the ViralSEQ™ Lentivirus Proviral DNA

TiterKit ..................................................................... 22

Prepare the Lentivirus DNA control serialdilutions ............................. 22

Extraction controlguidelines ................................................ 24

Prepare the extraction/ recovery control (ERC) for the ViralSEQ™ Lentivirus

Proviral DNA TiterKit ..................................................... 24

Triplicate extractions ....................................................... 25

ViralSEQ™ Lentivirus Titer Kits User Guide 3

■CHAPTER3Sample extraction with the PrepSEQ™ Nucleic Acid

Sample PreparationKit ........................................................... 26

Manual protocol for DNA/RNA extraction ......................................... 26

Digest the test samples and controls ......................................... 26

Bind theDNA/RNA ......................................................... 27

Wash theDNA/RNA ........................................................ 27

Elute theDNA/RNA ........................................................ 28

Automated protocol for DNA/RNA extraction ...................................... 29

Before each use of thekit ................................................... 29

Prepare the lysis plate ...................................................... 29

Process samples on theinstrument .......................................... 30

■CHAPTER4Set up, run, and review ViralSEQ™ Lentivirus Physical

Titer Kit experiments .............................................................. 31

Treat samples with DNase I, RNase‑free(1U/µL) ................................... 32

Prepare the kit reagents and premixsolution ...................................... 33

Prepare the PCR reactions ...................................................... 34

Setup, run, and analyze samples with AccuSEQ™ Software v3.1 on the

QuantStudio™ 5Instrument .................................................... 35

Sign in to the AccuSEQ™ Real‑Time PCR Software ............................. 35

Create a ViralSEQ™ template ................................................ 36

Edit the run method and optical ﬁlter selection ................................ 39

Assign plate and well attributes .............................................. 40

Load the plate in theinstrument ............................................. 43

Start the run (QuantStudio™ 5 Real‑Time PCR Instrument) ...................... 44

Monitor therun ............................................................ 45

Review the results .............................................................. 47

Calculate the titer(VP/mL) .................................................. 49

Analyze the data without AccuSEQ™ Real‑Time PCR Software .................. 49

Calculate lentivirus titers from qPCRdata ..................................... 50

■CHAPTER5Set up, run, and review ViralSEQ™ Lentivirus Proviral DNA

Titer Kit experiments .............................................................. 51

Prepare the kit reagents and premixsolution ...................................... 52

Prepare the PCR reactions ...................................................... 52

Setup, run, and analyze samples with AccuSEQ™ Software v3.1 on the

QuantStudio™ 5Instrument .................................................... 54

Sign in to the AccuSEQ™ Real‑Time PCR Software ............................. 54

Create a ViralSEQ™ template ................................................ 55

Edit the run method and optical ﬁlter selection ................................ 58

Assign plate and well attributes .............................................. 59

Load the plate in theinstrument ............................................. 62

Contents

4ViralSEQ™ Lentivirus Titer Kits User Guide

Start the run (QuantStudio™ 5 Real‑Time PCR Instrument) ...................... 63

Monitor therun ............................................................ 64

Review the results .............................................................. 66

Calculate the infectious titer ................................................. 68

Analyze the data without AccuSEQ™ Real‑Time PCR Software .................. 68

Calculate provirus copies and lentivirus titers from qPCRdata ................... 69

■APPENDIXATroubleshooting .................................................... 70

PrepSEQ™ sample preparation ................................................... 70

ViralSEQ™assays .............................................................. 70

■APPENDIXBUse the ViralSEQ™ Lentivirus Physical Titer Kit with the

7500 Fast Real‑Time PCR Instrument and AccuSEQ™ softwarev2.x ........ 73

Required materials notsupplied ................................................. 73

Plate document:settings ........................................................ 74

Create a plate document in the AccuSEQ™ software ................................ 74

Run the plate .................................................................. 76

Analyze the results ............................................................. 77

Calculate lentivirus titers from qPCRdata ......................................... 78

■APPENDIXCUse the ViralSEQ™ Lentivirus Proviral DNA Titer Kit with

the 7500 Fast Real‑Time PCR Instrument and AccuSEQ™ softwarev2.x .... 79

Required materials notsupplied ................................................. 79

Plate document:settings ........................................................ 80

Create a plate document in the AccuSEQ™ software ................................ 80

Run the plate .................................................................. 82

Analyze the results ............................................................. 83

Calculate provirus copies and lentivirus titers from qPCRdata ....................... 83

■APPENDIXDGood laboratory practices ........................................ 85

Good laboratory practices for PCR and RT-PCR ................................... 85

Good pipetting practices ........................................................ 85

Avoiding false positives due to cross-contamination ................................ 86

Plate layoutsuggestions ........................................................ 86

■APPENDIXESafety ............................................................... 87

Chemicalsafety ................................................................ 88

Biological hazardsafety ......................................................... 89

Contents

ViralSEQ™ Lentivirus Titer Kits User Guide 5

■APPENDIXFDocumentation and support ...................................... 90

Relateddocumentation ......................................................... 90

Customer and technical support ................................................. 90

Limited product warranty ........................................................ 91

Contents

6ViralSEQ™ Lentivirus Titer Kits User Guide

Product information

■Productdescription .................................................................... 7

■Contents and storage .................................................................. 8

■Required materials notsupplied ........................................................ 10

■Lentivirus production ................................................................. 13

■Kit controls .......................................................................... 13

■Workﬂow: ViralSEQ™ Lentivirus Physical TiterKit ......................................... 15

■Workﬂow: ViralSEQ™ Lentivirus Proviral DNA TiterKit ..................................... 16

Product description

The Applied Biosystems™ ViralSEQ™ Lentivirus Titer Kits are TaqMan™-based, real-time PCR assays

for process development and characterization of lentivirus vectors. The ViralSEQ™ Lentivirus Physical

Titer Kit quantitates the amount of lentivirus in the supernatants of cell-based, bioproduction systems.

The ViralSEQ™ Lentivirus Proviral DNA Titer Kit quantitates genome copies integrated into cells post-

transduction. Both kits use provided controls to generate standard curves used in quantitation. With

speciﬁc quantitation of viral and proviral copy numbers, qPCR data from physical and infectious titers

can be easily compared. Together, the ViralSEQ™ Lentivirus Titer Kits provide a convenient method to

correlate total and infectious lentiviral particles during process development and optimization.

Both the ViralSEQ™ Lentivirus Physical Titer Kit and the ViralSEQ™ Lentivirus Proviral DNA Titer Kit

are compatible with the PrepSEQ™ Nucleic Acid Sample Preparation Kit (Cat.A50485), which oers

both manual and automated sample preparation workﬂows. For real‑time PCR, both kits have been

validated on the Applied Biosystems™ 7500 Fast Real-Time PCR System and the Applied Biosystems™

QuantStudio™ 5 Real‑Time PCR System. Data analysis is streamlined using AccuSEQ™ Real‑Time

PCR Software that provides accurate quantitation. The software has security, audit, and e-signature

capabilities to enable 21 CFR Pt 11 compliance.

•The ViralSEQ™ Lentivirus Physical Titer Kit can be used to quantitate viral titers through the

measurement of lentivirus RNA in the supernatants of cell-based production systems.

•The ViralSEQ™ Lentivirus Proviral DNA Titer Kit is used to quantitate the number of proviral copies

incorporated into host cells following transduction.

1

ViralSEQ™ Lentivirus Titer Kits User Guide 7

Contents and storage

ViralSEQ™ Lentivirus Titer Kits

Table1ViralSEQ™ Lentivirus Physical Titer Kit (Cat.No.A52597)

Contents Cap color Amount Storage

Box 1, ViralSEQ™ Lentivirus Physical Titer qPCR Reagents

Physical Titer Assay Mix

(Gold)

275µL

Protect from light.

–25°C to –15°C

25✕ RT-PCR Enzyme Mix

(Green)

110µL

–30°C to –10°C

2✕ RT-PCR Buer

(Red)

1.375mL

Nuclease Free Water

(White)

1.0mL –25°C to –15°C until ﬁrst use. After

ﬁrst use, store at 2–8°C.

Box 2, ViralSEQ™ Lentivirus Physical Titer RNA Control

Physical Titer RNA Control (2 ✕1010

copies/µL)

(Gray)

44µL

–25°C to –15°C

RNA Dilution Buer Clear bottle 7.0mL

Table2ViralSEQ™ Lentivirus Physical Titer Kit with PrepSEQ™ Nucleic Acid Sample Preparation Kit

(Cat.No.A52598)

Contents Cat. No. Amount

ViralSEQ™ Lentivirus Physical Titer Kit A52597 1 kit

PrepSEQ™ Nucleic Acid Sample Preparation Kit A50485 1 kit

DNase I, RNase‑free (1U/µL) EN0521 1 kit

Chapter1Product information

Contents and storage

1

8ViralSEQ™ Lentivirus Titer Kits User Guide

Table3ViralSEQ™ Lentivirus Proviral DNA Titer Kit (Cat.No.A53561)

Contents Cap color Amount Storage

Box 1, ViralSEQ™ Lentivirus Proviral DNA Titer qPCR Reagents

Proviral DNA Titer Assay Mix

(Purple)

660µL

Protect from light.

–25°C to –15°C

2✕ Environmental Master Mix 2.0

(Orange)

2 ✕0.75mL –25°C to –15°C

Nuclease Free Water

(White)

1.0mL –25°C to –15°C until ﬁrst use. After

ﬁrst use, store at 2–8°C.

Box 2, ViralSEQ™ Lentivirus Proviral DNA Titer Control

Proviral DNA Titer Control (2✕108

copies/µL)

(Blue) 44µL

–25°C to –15°C

IMPORTANT! Do not store below

–25°C.

DNA Dilution Buer Clear bottle 7.0mL –25°C to –15°C

Table4ViralSEQ™ Lentivirus Proviral DNA Titer Kit with PrepSEQ™ Nucleic Acid Sample Preparation

Kit (Cat.No.A53562)

Contents Cat. No. Amount

ViralSEQ™ Lentivirus Proviral DNA Titer Kit A53561 1 kit

PrepSEQ™ Nucleic Acid Sample Preparation Kit A50485 1 kit

PrepSEQ™ Nucleic Acid Sample Preparation Kit (included with Cat.Nos.A52598

and A53562)

Table5PrepSEQ™ Nucleic Acid Sample Preparation Kit

Contents Amount Storage

Box 1, PrepSEQ™ Nucleic Acid Extraction Kit

Lysis Buer 2×50mL

Room temperature

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.

50mL

Chapter1Product information

Contents and storage 1

ViralSEQ™ Lentivirus Titer Kits User Guide 9

Table 5 PrepSEQ Nucleic Acid Sample Preparation Kit(continued)

Contents Amount Storage

ProteinaseK (PK) BuerII[1]

Recommended for new manual protocols.

Required for automated protocols.

Room temperature11mL

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

–20°C or below

Yeast tRNA, 10mg/mL 0.5mL

Glycogen, 5mg/mL 2×1.0mL

[1] Also sold separately (Cat.No.4415320).

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.

Catalog numbers that appear as links open the web pages for those products.

Required for the ViralSEQ™ Lentivirus Titer Kits

Item Source

Real-time PCR Instrument and software

QuantStudio™ 5 Real‑Time PCR System with AccuSEQ™ software v3.1

or later

Contact your local sales

representative.

Equipment

(If using automated sample preparation) KingFisher™ Flex Puriﬁcation

System with 96 Deep-Well Head

5400630

Microcentrifuge MLS

Laboratory mixer, vortex, or equivalent MLS

Plate centrifuge MLS

VeritiPro™ 96-well Thermal Cycler A48141

Chapter1Product information

Required materials not supplied

1

10 ViralSEQ™ Lentivirus Titer Kits User Guide

(continued)

Item Source

Consumables

Disposable gloves MLS

Aerosol-resistant pipette tips MLS

Nuclease-free microcentrifuge tubes MLS

Pipettors:

•Positive-displacement

•Air-displacement

•Multichannel

MLS

MicroAmp™ Optical 96-Well Reaction Plate, 0.2 mL 4316813

Consumables for the QuantStudio™ 5 Real‑Time PCR System

MicroAmp™ Fast Optical 96‑Well Reaction Plate with Barcode, 0.1 mL 4346906

MicroAmp™ 96-Well Tray/Retainer Set 403081

MicroAmp™ Optical Adhesive Film 4360954

MicroAmp™ 96-Well Base N8010531

MicroAmp™ Adhesive Film Applicator 4333183

Recommended for sample extraction

PrepSEQ™ Nucleic Acid Sample Preparation Kit[1] A50485

Recommended for DNase treatment (ViralSEQ™ Lentivirus Physical Titer Kit only)

DNase I, RNase‑free (1U/µL)[2] EN0521

[1] Included with Cat.Nos.A52598 and A53562.

[2] Included with Cat.No.A52598.

Required for manual sample preparation

Item Source

Equipment

Magnetic stand, 16-position 12321D

Block heater for use with 2‑mL tubes.

Manual DNA/RNA extraction involves two incubations at dierent

settings, so two heaters may be convenient.

MLS

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

Vortex MLS

Chapter1Product information

Required materials not supplied 1

ViralSEQ™ Lentivirus Titer Kits User Guide 11

Item Source

Vortex Adapter-60, for use with the Vortex-Genie™AM10014

Consumables

Disposable gloves MLS

Aerosol-resistant micropipette tips MLS

Pipettors, P1000, P200, P20 and P10:

•Positive-displacement

•Air-displacement

•Multichannel

MLS

Nonstick, RNase-free Microfuge Tubes, 1.5‑mL (1 box; 250 tubes/box) AM12450

Safe-Lock Tube, 2.0‑mL VWR™ 62111-754

Reagents

Ethanol, 95%

IMPORTANT! Do not use denatured ethanol. It contains components

that are not compatible with the protocol.

MLS

Isopropanol, 100% MLS

1X PBS (free of Mg and Ca) MLS

Required for automated sample preparation

Table6Pharma KingFisher™ Flex Puriﬁcation System with 96 Deep-Well Head (Cat.No.A31508)

accessories

Item Source

Pharma KingFisher™ Flex 96 Deep-Well Tip Combs A43074

Pharma KingFisher™ Flex 96 Deep-Well Plates A43075

Pharma KingFisher™ Flex Magnetic Head for 96 Deep-Well Plate A31542

Pharma MagMAX™ 96 PCR Well Magnetic Head 4472991

Pharma KingFisher™ Flex 96 Standard Plate A43076

(Optional) Pharma Magnetic Stand-96 A31543

Chapter1Product information

Required materials not supplied

1

12 ViralSEQ™ Lentivirus Titer Kits User Guide

Table7Additional materials

Item Source

Consumables

Disposable gloves MLS

Aerosol-resistant micropipette tips MLS

Pipettors, P1000, P200, P20 and P10:

•Positive-displacement

•Air-displacement

•Multichannel

MLS

Nonstick, RNase-free Microfuge Tubes, 1.5‑mL (1 box; 250 tubes/box) AM12450

Reagents

Ethanol, 95%

IMPORTANT! Do not use denatured ethanol. It contains components

that are not compatible with the protocol.

MLS

Isopropanol, 100% MLS

1X PBS (free of Mg and Ca) MLS

Lentivirus production

The ViralSEQ™ Lentivirus Physical Titer Kit is used to quantitate viral particles produced using cell-

based systems. We recommend using the LV-MAX™ Lentiviral Production System (Cat.No. A35684) to

produce high titer lentiviral vectors. The system is based on the transient transfection of high-density,

suspension HEK293F cells adapted to a chemically deﬁned, serum-free and protein free medium. For

more information, see the LV-MAX™ Lentiviral Production System User Guide (Pub.No.MAN0017000).

Kit controls

Table8ViralSEQ™ Lentivirus Physical Titer Kit

Control Use

Internal Positive Control (IPC)

(Included in the Physical Titer Assay Mix)

Lack of ampliﬁcation Indicates PCR inhibition.

Physical Titer RNA Control (2 ✕1010 copies/µL) Ampliﬁcation indicates that the PCR was successful.

Negative control (RNA Dilution Buer) Ampliﬁcation indicates contamination.

Chapter1Product information

Lentivirus production 1

ViralSEQ™ Lentivirus Titer Kits User Guide 13

Table9ViralSEQ™ Lentivirus Proviral DNA Titer Kit

Control Use

Internal Positive Control (IPC)

(Included in the Proviral DNA Titer Assay Mix)

Lack of ampliﬁcation Indicates PCR inhibition.

Proviral DNA Titer Control (2✕108 copies/µL) Ampliﬁcation indicates that the PCR was successful.

Negative control (DNA Dilution Buer) Ampliﬁcation indicates contamination.

Chapter1Product information

Kit controls

1

14 ViralSEQ™ Lentivirus Titer Kits User Guide

Workﬂow: ViralSEQ™ Lentivirus Physical Titer Kit

ViralSEQ™ Lentivirus Physical Titer Kit Workﬂow

Produce lentivirus with the

LV-MAX™ Lentiviral Production

System

varies

Prepare reagents and serial

dilutions

1h

Sample extraction with the

PrepSEQ™ Nucleic Acid Sample

Preparation Kit

1h30min

Treat samples with DNase I,

RNase‑free (1U/µL)

45min

Prepare the PCR reactions

15min

Start the run (QuantStudio™ 5

Real‑Time PCR Instrument)

2h

Review the results

20min

Chapter1Product information

Workﬂow: ViralSEQ™ Lentivirus Physical Titer Kit 1

ViralSEQ™ Lentivirus Titer Kits User Guide 15

Workﬂow: ViralSEQ™ Lentivirus Proviral DNA Titer Kit

ViralSEQ™ Lentivirus Proviral DNA Titer Kit Workﬂow

Lentivirus transduction of target

cells

varies

Prepare reagents and serial

dilutions

1h

Sample extraction with the

PrepSEQ™ Nucleic Acid Sample

Preparation Kit

1h30min

Prepare the PCR reactions

15min

Start the run (QuantStudio™ 5

Real‑Time PCR Instrument)

2h

Review the results

20min

Chapter1Product information

Workﬂow: ViralSEQ™ Lentivirus Proviral DNA Titer Kit

1

16 ViralSEQ™ Lentivirus Titer Kits User Guide

Prepare reagents and serial dilutions

■Prepare the PrepSEQ™ reagents: before ﬁrst use of thekit ................................ 17

■Prepare PrepSEQ™ reagents: before each use of thekit ................................... 18

■Guidelines for optimalyields ........................................................... 19

■Serial dilutions and sample preparation for the ViralSEQ™ Lentivirus Physical TiterKit ........ 19

■Serial dilutions and sample preparation for the ViralSEQ™ Lentivirus Proviral DNA TiterKit ..... 22

We recommend using the PrepSEQ™ Nucleic Acid Sample Preparation Kit (Cat.No.A50485) for sample

preparation. The kit uses chemical lysis and magnetic beads to extract nucleic acid from recombinant

lentiviruses and integrated proviruses.

Prepare the PrepSEQ™ reagents: before ﬁrst use of the kit

Magnetic beads

1. Set a block heater to 37°C.

2. Incubate the Magnetic Particle suspension at 37°C for a minimum of 10 minutes with intermittent

vortexing at 900 rpm, or until the particles are completely suspended.

Binding Solution

1. Add 45 mL of 100% isopropanol to the Binding Solution bottle.

2. Label the bottle to indicate that it contains isopropanol, then store the bottle at room temperature.

Wash Buer Concentrate

1. Add 74 mL of 95% ethanol to one bottle of PrepSEQ™ Wash Buer Concentrate, then mix

completely.

2. Label the bottle to indicate that it contains ethanol, then store the bottle at room temperature.

2

ViralSEQ™ Lentivirus Titer Kits User Guide 17

Prepare PrepSEQ™ reagents: before each use of the kit

Proteinase K (PK) mix

•Use ProteinaseK (PK) BuerII for all protocols.

Note: Proteinase K (PK) Buer is provided in the kit for use by laboratories that had previously

validated with this buer. If required, use exactly as described for ProteinaseK (PK) BuerII.

•Prepare a fresh mix before each use of the kit.

•Include a 10% overage to account for pipetting losses.

Component

Number of extractions

1 7 10 13 25

Proteinase K, 20mg/mL 10μL 70μL 100μL 130μL 250μL

ProteinaseK (PK) BuerII 60μL 420μL 600μL 780μL 1,500μL

Lysis solution

•Prepare a fresh mixture immediately before use or during Proteinase K incubation.

•Prepare 360 µL (amount required) of lysis solution mix per sample.

Reagent Volume for 1 extraction Volume for ~20 extractions

Glycogen, 5mg/mL 8.32µL 180µL

Yeast tRNA, 10mg/mL 0.18µL 4µL

Lysis Buer 351.5µL 7,600µL

Total 360µL 7,784µL

Chapter2Prepare reagents and serial dilutions

Prepare PrepSEQ™ reagents: before each use of the kit

2

18 ViralSEQ™ Lentivirus Titer Kits User Guide

Guidelines for optimal yields

•Maintain a homogenous suspension of the magnetic beads to maximize the surface area to which

the DNA/RNA can bind. The appearance of the mixture should be homogenous after mixing.

•After drying, the DNA/RNA remains bound to the magnetic beads. Do not allow the magnetic

beads to over-dry because this reduces the elution eciency; over-dried beads are not easily

resuspended.

•During manual elution, vortex every 2 minutes to assist elution. This will result in better yield during

recovery.

Note: Some test samples cause the beads to adhere very ﬁrmly to the tube wall, while others form

loose pellets that detach during the vortex steps. All pellets should dissolve with vortexing during

heated elution. If vortexing does not result in full resuspension, then wash the beads o the tube by

pipetting.

Note: White or brown precipitate may form in the Magnetic Particles tube if it is stored at 2–8°C.

The precipitate will dissolve when it is heated to 37°C for a minimum of 10 minutes with intermittent

vortexing. Make sure the precipitate is completely dissolved before using the beads.

Serial dilutions and sample preparation for the ViralSEQ™

Lentivirus Physical Titer Kit

Collect lentivirus particles for RNA extraction and sample preparation. For test samples that contain

levels of RNA above the highest point of the assay standard curve (109 copies per reaction), dilute these

samples (from 1:100 up to 1:10,000) with a solution of 1 X PBS (free of Mg and Ca) or 50 mM Tris, pH

8.0, 0.5 M NaCl.

Note: Use sample dilution buer as the negative extraction control or dilute extracted RNA with elution

buer before running the PCR reaction. Diluting samples in water or TE reduces extraction eciency.

Prepare the Lentivirus RNA control serial dilutions

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.

Chapter2Prepare reagents and serial dilutions

Guidelines for optimal yields 2

ViralSEQ™ Lentivirus Titer Kits User Guide 19

Prepare the serial dilutions

Thaw the Physical Titer RNA Control (2 ✕1010 copies/µL) on ice. Vortex at medium speed for 5

seconds, brieﬂy centrifuge, then place on ice until use.

1. Label nonstick 1.5‑mL microfuge tubes: NTC, SD1, SD2, SD3, SD4, SD5, and SD6 [used for limit

of detection (LOD)].

2. Add 35µL of RNA Dilution Buer (RDB) to the NTC (no template control) tube. Place the tube on

ice.

3. Perform the serial dilutions.

When dispensing RNA, pipette up and down gently. After each transfer, vortex for 7 seconds, then

centrifuge brieﬂy.

Table10Standard curve dilutions (ViralSEQ™ Lentivirus Physical Titer Kit)

Serial dilution (SD)

tube Dilution Dilution

factor

Copies/ PCR reaction

(5μL of diluted RNA)

SD1 5μL RNA control + 495μL RDB 100 1.00E+09

SD2 5μL SD1 + 495μL RDB 100 1.00E+07

SD3 5μL SD2 + 495μL RDB 100 1.00E+05

SD4 5μL SD3 + 495μL RDB 100 1,000

SD5 10μL SD4 + 190μL RDB 20 50

SD6 (LOD) 40μL SD5 + 160μL RDB 5 10

Store the standard curve dilution tubes at 4°C or on ice. Use the dilutions within 6 hours for RT-qPCR.

Extraction control guidelines

We recommend that you use the following extraction controls:

Type of control Contains Number to run Used to

Negative (NEG)[1] 1X PBS (free of Mg and Ca) 1 per batch of

extractions

To monitor for contamination of the

extraction reagents.

Extraction/recovery (ERC) Positive control from the kit 3 per sample •Evaluate the eciency of DNA

or RNA extraction, recovery,

and quantiﬁcation from test

samples.

•Verify reagent and system

performance.

[1] Optional during routine testing.

Chapter2Prepare reagents and serial dilutions

Serial dilutions and sample preparation for the ViralSEQ™ Lentivirus Physical Titer Kit

2

20 ViralSEQ™ Lentivirus Titer Kits User Guide

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Thermo Fisher Scientific ViralSEQ Lentivirus Titer Kits User guide

Thermo Fisher Scientific ViralSEQ Lentivirus Titer Kits User guide

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