PRODUCT INFORMATION SHEET

SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA

Polymerase
Catalog Numbers 12574-018 and 12574-026
Doc. Part No. 12574.pps Pub. No. MAN0001094 Rev. A.0

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Product description
The Invitrogen™ SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is designed for the sensitive, reproducible, end-
point detection and analysis of RNA molecules by RT–PCR. Using this convenient one-step formulation, you can perform both cDNA synthesis
and PCR amplification in a single tube using gene-specific primers, and target RNAs from either total RNA or mRNA. The system uses a mixture
of SuperScript™ III Reverse Transcriptase and Platinum™ Taq DNA Polymerase in an optimized reaction buffer, and it can detect a wide range of
RNA targets, from 200 bp to 4.5 kb. The amount of starting material can range from 0.01 pg to 1 μg of total RNA.
SuperScript™ III Reverse Transcriptase is a version of M–MLV RT that has been engineered to reduce RNase H activity and provide increased
thermal stability. The enzyme can synthesize cDNA at a temperature range of 45–60°C, providing increased specificity, higher yields of cDNA, and
more full-length product than other reverse transcriptases. Because SuperScript™ III RT is not significantly inhibited by ribosomal and transfer
RNA, it can be used to synthesize cDNA from total RNA.
Platinum™ Taq DNA Polymerase is recombinant Taq DNA polymerase complexed with a proprietary antibody that blocks polymerase activity at
ambient temperatures. Activity is restored after the denaturation step in PCR cycling at 94°C, providing an automatic “hot start” in PCR for
increased sensitivity, specificity, and yield.
The 2X Reaction Mix included in the kit consists of a proprietary buffer system that has been optimized for reverse transcription and PCR, Mg2+,
dNTPs, and stabilizers. The convenient 2X format allows you to add template and primer at any desired concentration. A tube of 5 mM MgSO4 is
included in the kit for further optimization of the Mg2+ concentration. Sufficient reagents are provided for 25 or 100 amplification reactions of 50 μL
each.
Note: This kit has been optimized for end-point RT–PCR. For quantitative real-time RT–PCR, use the SuperScript™ III Platinum™ One–Step
Quantitative RT–PCR System (see “Ordering information” on page 3).

Contents and storage

Cat. No. 12574-018 Cat. No. 12574-026
Contents Storage
(25 reactions) (100 reactions)
SuperScript™ III RT/Platinum™ Taq Mix 50 μL 200 μL
2X Reaction Mix (a buffer containing 0.4 mM of each dNTP, 3.2 mM Store all components
1 mL 3 × 1 mL
MgSO4) at −30°C to −10°C.
5 mM Magnesium Sulfate 500 μL 500 μL

Procedural guidelines
Guidelines for RNA
• High-quality, intact RNA is essential for successful full-length cDNA synthesis.
• For low copy-number genes or longer targets, use more starting material (>10 ng total RNA).
• RNA should be devoid of any RNase contamination and aseptic conditions should be maintained.
• We recommend TRIzol™ Reagent for isolation of total RNA. See “Ordering information” on page 3. Oligo(dT) selection for poly(A)+ RNA is
typically not necessary, although it may improve the yield of specific cDNAs.

Guidelines for primers

• We recommend using gene-specific primers (GSPs). We do not recommend using oligo(dT) or random primers, because they can generate
nonspecific products in the one-step procedure and the amount of RT-PCR product may be reduced.
• A final primer concentration of 0.2 μM for each primer is generally optimal. However, for best results, we recommend performing a primer
titration of 0.15–0.5 μM.
• Design primers that anneal to the mRNA sequence in exons on both sides of an intron or exon/exon boundary, to allow differentiation between
the amplified cDNA and potential contaminating genomic DNA.
• Primers should not be self-complementary or complementary to each other at the 3¢ ends.

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

Guidelines for magnesium and dNTP concentration
• MgSO4 is included in the 2X Reaction Mix at a final concentration of 1.6 mM, which works well for most targets. If needed, the magnesium
concentration can further be optimized (usually between 1.4 and 2 mM) with the 5 mM MgSO4 provided in the kit.
• dNTPs are included in the 2X Reaction Mix at a final concentration of 200 μM, which is optimal for most reactions.

Guidelines for PCR

• Program the thermal cycler before setting up the reaction. The thermal cycler should be preheated to 45–60°C, depending on the temperature
selected for cDNA synthesis.
• For difficult or high GC-content templates, use a 55–60°C cDNA synthesis temperature.
• Keep all components, reaction mixes, and samples on ice. After preparation of the samples, transfer them to the preheated thermal cycler and
immediately start the RT-PCR program.
• Efficient cDNA synthesis can be accomplished in a 15–30-minute incubation at 45–60°C. For small targets, an incubation time of 5 minutes may
be sufficient.
• SuperScript™ III RT is inactivated, Platinum™ Taq DNA Polymerase is reactivated, and the RNA/cDNA hybrid is denatured during the 2-minute
incubation at 94°C.
• The annealing temperature should be 10°C below the melting temperature of the primers used.
• The extension time varies with the size of the amplicon (approximately 1 minute per 1 kb of amplicon).
• For all targets up to 4.5 kb, 2 μL of SuperScript™ III RT/Platinum™ Taq Mix is sufficient.

Methods ™ ™
The following cycling conditions were established and tested using a GeneAmp PCR System 9600 and 2400 and a DNA Engine Opticon PTC-200.
You may need to adjust these conditions for other thermal cyclers. Efficient cDNA synthesis can be achieved in a 15–30-minute incubation at
45−60°C. We recommend a 30-minute incubation at 55°C as a general starting point. The optimal temperature for reverse transcription depends on
primer and target sequences. Cycling conditions may have to be further optimized for different sequences. Three-step cycling (separate annealing
and extension steps) is required.
1. Program the thermal cycler so that cDNA synthesis is followed immediately by PCR amplification, as follows:
Final extention
cDNA synthesis and pre-denaturation Denature Anneal Extend
(optional)
1 CYCLE 40 CYCLES 1 CYCLE
45–60°C 94°C 94°C 55–66°C 68°C 68°C
15–30 minutes 2 minutes 15 seconds 30 seconds 1 minute/kb 5 minutes
2. Add the following to a 0.2–mL, nuclease-free, thin-walled PCR tube on ice. For multiple reactions, you can prepare a master mix to minimize
reagent loss and enable accurate pipetting.
Component Volume
2X Reaction Mix 25 μL
Template RNA (.01 pg to 1 μg) x μL
Sense primer (10 μM) 1 μL
Anti-sense primer (10 μM) 1 μL
SuperScript™ III RT/Platinum™ Taq Mix[1] 2 μL
Autoclaved distilled water to 50 μL
[1] You can verify the absence of genomic DNA in RNA preparations by omitting the SuperScript™ III RT/Platinum™ Taq Mix and substituting 2 units of Platinum™ Taq DNA
Polymerase in the reaction.
3. Gently mix and make sure that all the components are at the bottom of the amplification tube. Centrifuge briefly if needed. Depending on the
thermal cycler used, overlay with silicone oil if necessary.
4. Place the reaction in the preheated thermal cycler programmed as described above. Collect the data and analyze the results.

2 SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase Product Information Sheet
Troubleshooting
Observation Possible cause Recommended action
No amplificiation product No cDNA synthesis (temperature too For the cDNA synthesis step, incubate <55°C.
high)
RNase contamination Maintain aseptic conditions; add RNase inhibitor.
Not enough starting template RNA Increase the concentration of template RNA; use 100 ng to 1 μg of total
RNA.
RNA has been damaged or degraded Replace RNA if necessary.
RT inhibitors are present in RNA Remove inhibitors in the RNA preparation by an additional 70% ethanol
wash.
Note: Inhibitors of RT include SDS, EDTA, guanidium salts, formamide,
sodium phosphate, and spermidine.
Annealing temperature is too high Decrease temperature as necessary.
Extension time is too short Set extension time for at least 60 seconds per kb of target length.
Cycle number is too low Increase cycle number.
Low specificity Reaction conditions not optimal Optimize magnesium concentration.
Optimize the primer.
Optimize the annealing temperature and extension time.
Increase temperature of RT reaction to 60°C.
Oligo(dT) or random primers used for Use only gene-specific primers.
first-strand synthesis
Unexpected bands after Contamination by genomic DNA Pretreat RNA with DNase I, Amplification Grade (Cat. No. 18068-015), as
electrophoretic analysis described in the DNase I documentation.
Design primers that anneal to sequence in exons on both sides of an
intron or at the exon/exon boundary of the mRNA to differentiate
between amplified cDNA and potential contaminating genomic DNA.
To test if products were derived from DNA, substitute 2 units of
Platinum™ Taq DNA Polymerase for the SuperScript™ III RT/Platinum™
Taq Mix in the reaction.
Nonspecific annealing of primers Vary the annealing temperature.
Optimize the magnesium concentration for each template and primer
combination.
Primers formed dimers Design primers without complementary sequences at the 3' ends.

Ordering information
The following products are also available. Unless otherwise indicated, all materials are available through thermofisher.com.
Item Amount Source
SuperScript™ III Platinum™ One-Step Quantitative RT-PCR System 100 reactions 11732-020
500 reactions 11732-088
TRIzol™ Reagent 100 mL 15596-026
200 mL 15596-018
DNase I, Amplification Grade 100 units 18068-015
Custom Primers To order, visit thermofisher.com

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SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase Product Information Sheet 3
The information in this guide is subject to change without notice.
DISCLAIMER
TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES 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. MAN0001094
Revision Date Description
A.0 28 April 2016 Format, style, and legal updates
— 18 November 2011 Baseline for this revision history

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
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