qScript® XLT One-Step RT-PCR Kit
Cat. No

95143-020
95143-200

Size:

20 x 25-µL reactions
200 x 25-µL reactions

Store at -25°C to -15°CDescription
The qScript XLT One-Step RT-PCR Kit is a convenient and highly sensitive system for amplification of RNA templates up to 2 kb. cDNA synthesis and PCRamplification are carried out in the same tube without opening between procedures. This system has been optimized to deliver maximum RT-PCR efficiency,sensitivity, and specificity.
qScript XLT is an engineered M-MLV reverse transcriptase with reduced RNase H activity and improved activity and stability at higher temperatures. The
use of higher temperatures (48 to 55°C) for the cDNA synthesis step of one-step RT-PCR provides higher specificity for primer annealing and disruption of
RNA secondary structure that can interfere with cDNA synthesis. The enzyme is supplied as a mixture with ribonuclease inhibitor protein to protect theintegrity of RNA templates in crude lysates or samples where RNase contamination may limit assay sensitivity.
A key component of the system is the One-Step ToughMix®. This master mix is highly resistant to PCR inhibitors and contains an ultra pure, hot-start, highly
processive thermostable DNA polymerase that is blended with a proof-reading (3’-exonuclease) polymerase for improved PCR fidelity and fragment lengths.
High-avidity monoclonal antibodies provide an extremely stringent automatic hot-start that minimizes the potential for primer-dimer and other non-specific
PCR artifacts without compromising polymerase activities. Highly specific amplification is crucial to successful RT-PCR as non-specific product(s) cancompete with amplification of the target sequence and impair PCR efficiency. The proprietary reaction buffer has been specifically formulated to maximize
activities of both the reverse transcriptase and thermostable DNA polymerase while minimizing the potential for primer-dimer and other non-specific PCR
artifacts.
GelTrack® Loading Dye is a mixture of blue and yellow electrophoresis-tracking dyes that migrate at approximately 4kb and 50 bp. This optional
component simplifies post PCR analysis, allowing direct loading of RT-PCR product on agarose gels following amplification.
The GelTrack Loading Dye solution is not included with the sample size 95143-020

Components
Reagent
qScript XLT One-Step Reverse
Transcriptase (25X)
One-Step ToughMix (2X)
GelTrack Loading Dye (50X)
Nuclease-free water

Description
25X concentrated mixture of qScript XLT reverse transcriptase and recombinantribonuclease inhibitor protein.
2X concentrated reaction buffer containing dNTPs, magnesium chloride, hot-start
DNA polymerase, and stabilizers
50X concentrated mixture of RT-PCR compatible, blue and yellow electrophoresistracking dyes

Quantity
1 x 200 µL
2 x 1.25 mL
1 x 0.4 mL
2 x 1.5 mLStorage and Stability
Store components in a constant temperature freezer at -25°C to -15°C upon receipt.
Repeated freezing and thawing of the ToughMix will not affect product performance.
For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.
Guidelines for One-Step RT-PCR






Thaw components at room temperature. Mix thoroughly by gently vortexing, and then centrifuge to collect contents to the bottom of the tube before
using. Place all components on ice after thawing.
To maximize specificity, reactions should be assembled on ice. The thermostable DNA polymerase is inactive prior to high temperature activation;
however, qScript XLT reverse transcriptase is active at lower temperatures. During reaction assembly, equilibrate the thermocycler block to 50°C
(or your preferred temperature for cDNA synthesis) and pause the run. Transfer fully assembled reactions from ice to the pre-warmed thermocycler
and resume the run.
First-strand synthesis can be carried out between 42°C and 55°C. cDNA conversion of short fragments (under 500 nt) is complete within 5 to 10minutes. As a general starting point, use a 20-minute incubation at 48°C. We recommend a 3 minute incubation at 94°C to fully inactivate the RT
prior to PCR cycling.
Perform a minus RT control to verify that amplification product is derived from RNA sequence and not genomic DNA.

95143 / IFU-099.1 REV 04

1

Guidelines for One-Step RT-PCR Continued:Primer concentrations may need to be optimized for different target sequences. qScript XLT reverse transcriptase generally prefers 200 to 600 nM
primer for effective priming of the cDNA synthesis step. We recommend using 400 nM each primer as a general starting point. The optimal
concentration of each primer may need to be empirically determined. We suggest testing a range between 100 and 800 nM. Lower primer
concentrations (150 to 250 nM) often reduce non-specific products, should they occur. Higher primer concentrations (400 to 500 nM) can be used
to drive higher product yields for longer fragments.
Suggested input quantities of template are: 1 pg to 1 μg total RNA; 10 fg to 100 ng poly A(+) RNA; 10 to 1x108 copies virus RNA.
When performing multiple reactions, always prepare a reaction cocktail that contains all components except RNA template. After mixing, dispense
the reaction cocktail (15 to 20-µL volume / reaction) to each tube and then add the RNA template (5 to 10-µL volume / reaction) as a final component.
Allow for extra reactions (5-10%) to accommodate pipetting steps when assembling the reaction cocktail.
Thoroughly mix fully assembled reactions by gently vortexing, and then briefly centrifuge to collect contents.

Reaction Assembly (on ice)
Component
One-Step ToughMix (2X)
Forward primer (100 µM)
Reverse primer (100 µM)
GelTrack Loading Dyes (50X)
qScript XLT One-Step RT (25X)
Nuclease-free water
RNA template
Final Volume (μL)

Volume for
25-μL rxn.
12.5 µL
Variable (0.05 to 0.15 µL)
Variable (0.05 to 0.15 µL)
0.5 µL
1 µL
Variable
5 – 10 µL
25 µL

Reaction Cocktail
for 96 rxns (100)1250 µL
10 µL (for 400 nM)
10 µL (for 400 nM)
50 µL
100 µL
80 µL

Final Concentration
1X
200 – 600 nM
200 – 600 nM
1X (optional)
1X

variable
1,500 µL
Dispense 15 µL to each rxn.
Then add 10 µL RNA template
Note: For larger or smaller reaction volumes scale all components proportionally. Omit qScript XLT One-Step RT for minus RT control reactions.

Reaction Protocol
Incubate reactions as follows:
cDNA Synthesis
Initial denaturation
PCR cycling (30 - 45 cycles)
Denaturation:
Annealing:
Extension:

48°C, 20 min
94°C, 3 min
94°C, 10 to 20s
55 – 65°C, 20 to 60s
68ºC to 72°C, 1 minute per kb of target length

Quality Control
Kit components are free of contaminating DNase and RNase. The qScript XLT One-Step RT-PCR Kit is functionally tested in RT-PCR for amplification ofa 2-kb fragment of the APC gene from HeLa cell total RNA.

Limited Label Licenses
Use of this product signifies the agreement of any purchaser or user of the product to the following terms:
1. The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kit only.
Quantabio, LLC. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included
within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at www.quantabio.com. Some of theseadditional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by Quantabio, LLC. Quantabio,
LLC. neither guarantees them nor warrants that they do not infringe the rights of third-parties.
2. Other than expressly stated licenses, Quantabio, LLC. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.
3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. Quantabio, LLC. specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. Quantabio, LLC.
may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action
to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.
©2021 Quantabio, LLC. 100 Cummings Center Suite 407J Beverly, MA 01915; Telephone number: 1-888-959-5165.
Quantabio products are manufactured in Beverly, Massachusetts, Frederick, Maryland and Hilden, Germany.

Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
qScript, ToughMix and GelTrack are registered trademarks of Quantabio, LLC.

95143 / IFU-099.1 REV 04

2