Invitrogen™ SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase

Product Code.	No. of Reactions	unitSize
10779764	100 Reactions	100 reactions
10092922	25 Reactions	25 reactions

Product Code. 10779764 Supplier Invitrogen™ Supplier No. 12574035

Description

The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity is designed for convenient end-point detection and analysis of RNA molecules by one-step RT-PCR.

The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity is designed for convenient end-point detection and analysis of RNA molecules by one-step RT-PCR. This one-step formulation allows for both cDNA synthesis and PCR amplification to be performed in a single tube using gene-specific primers and target RNAs from either total RNA or mRNA. It also allows for the detection of a wide range of RNA targets (up to 10 kb in length) at variable concentrations (1 pg to 1 μg of total RNA). This system consists of two major components: SuperScript III RT/Platinum Taq High Fidelity enzyme mix and 2X reaction mix.

SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA and can be used to synthesize cDNA from total RNA. Platinum Taq DNA Polymerase High Fidelity is a an enzyme mixture composed of recombinant Taq DNA polymerase, Pyrococcus species GB–D polymerase, and Platinum Taq antibodies, which block polymerase activity at ambient temperatures enabling hot start PCR.

The 2X reaction mix consists of a proprietary buffer system optimized for reverse transcription and PCR amplification, Mg²⁺ optimized for universal use, deoxyribonucleotide triphosphates, and stabilizers.

The SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity offers:
• High-fidelity end-point detection and analysis of RNA molecules by RT-PCR
• PCR amplification and cDNA synthesis in a single tube
• Detection of a wide range of RNA targets from 300 bp to 10 kb
• Increased specificity with wide cDNA synthesis temperature range of 45−60°C

Note: For superior one-step RT-PCR performance, we recommend the SuperScript IV One-Step RT-PCR System (Cat. No. 12594025) or SuperScript IV One-Step RT-PCR System with ezDNase (Cat. No. 12595025). The SuperScript IV One-Step RT-PCR System combines the high processivity of SuperScript IV Reverse Transcriptase with the high-fidelity of Platinum SuperFi DNA Polymerase to provide unmatched product yields, specificity, and sensitivity in less time and for a broad target range, even with suboptimally pure RNA samples.

Order Info

Shipping Condition: Dry ice

Specifications

• Content And Storage:

  • SuperScript III RT/Platinum Taq High Fidelity Enzyme Mix (100 μL)
  • 2X Reaction Mix (3 x 1 mL)
  • 5 mM Magnesium Sulfate (500 μL)
  
  Store at –10 to –30°C.
  
  

• Detection Method: Gel Electrophoresis
• Format: One-Step RT-PCR System
• GC-Rich PCR Performance: High
• PCR Method: 1-step RT-PCR
• Polymerase: Platinum Taq High Fidelity
• Reaction Speed: 30 min.
• Technique: 1-Step RT-PCR
• Optimal Reaction Temperature: 50°C
• Quantity: 100 rxns
• Reverse Transcriptase: SuperScript III
• Ribonuclease H Activity: Reduced
• Shipping Condition: Dry Ice
• Final Product Type: PCR Amplified cDNA
• Hot Start: Built-In Hot Start
• No. of Reactions: 100 Reactions
• Reaction Format: Premixed Components
• Reagent Type: Reverse Transcription
• Size (Final Product): Up to 10 kb
• Starting Material: RNA

Frequently Asked Questions (FAQs)

How can I remove genomic DNA contamination from my sample prior to performing RT-PCR?

We recommend using ezDNase (Cat. No. 11766051). ezDNase Enzyme's high specificity for double-stranded DNA enables efficient and fast genomic DNA removal without reduction in the quality or quantity of RNA. ezDNase Enzyme is heat-labile and so can be easily deactivated by heat treatment at moderate temperature (55 degrees C). These features make ezDNase Enzyme an excellent choice for genomic DNA removal prior to reverse transcription reactions.

How much RNA should be employed for first-strand cDNA synthesis?

The amount of RNA template for a cDNA synthesis is highly flexible and depends upon the amount of sample available and an individual's need. In general, 1 µg total RNA is used in a typical 20-µL RT reaction.

Should I treat the cDNA with RNase H prior to downstream processing?

RNase H treatment is not always necessary. Many PCR reactions work without it. However, for cDNA synthesized with RNase H-deficient reverse transcriptases (like SuperScript II, III, and IV), RNA/cDNA hybrids—especially GC-rich ones—may not denature well, reducing PCR sensitivity. RNase H treatment can help in such cases. Additionally, RNase H treatment is beneficial for cloning larger fragments.

What percentage of RNA is converted to cDNA when performing reverse transcription?

This depends highly on the quality of the sample. mRNA itself makes up 1-5% of total RNA. Depending on the primer and enzyme used, reverse transcription can covert >70% of that into cDNA.

I'm setting up my RT reaction and am trying to decide whether I should use random primers, oligo(dT) primer, gene-specific primer, or oligo(dT)/random mix primers. What would you suggest?

Random primers are the best choice for degraded RNA, RNA with heavy secondary structure, non-polyadenylated RNA, or prokaryotic RNA. It is recommended only for two-step RT-PCR, and typically gives the highest yields, although the cDNA may not necessarily be full length. Oligo(dT) primers are good to use when trying to recover full-length cDNA from 2-step RT-PCR. The reaction is influenced by secondary structure and RNA quality. Gene specific primers should be used for very specific, mainly one-step RT-PCR reactions.

The DTT in my reverse transcription kit has precipitated—can I still use it?

No, the DTT will need to be replaced.

Are SuperScript II and III RTs RNase H minus?

These enzymes contain the domains of RNase H, but they have been mutated for reduced RNase H activity. In RNase H activity detection assays, we are not able to detect any RNase H activity.

Can I purchase the SuperScript III buffer separately?

Yes, we sell a M-MLV RT buffer (Cat. No. 18057018), which works with M-MLV RT, SuperScript II RT, and SuperScript III RT.

Will adding EDTA prior to heat-inactivation of DNase I inhibit reverse

EDTA chelates Mg2+ molecules on a 1:1 molar basis. If the amount of EDTA used for DNase I inactivation does not exceed the amount of Mg2+ present in the DNase reaction buffer, the resulting RNA solution can be used directly in the RT reaction. Otherwise, the sample should be purified to remove excess EDTA. Alternatively, consider using DNase removal methods that do not rely on EDTA or heat inactivation to avoid interference. To reduce the risk of compromised cDNA synthesis, we recommend performing gDNA removal with ezDNase Enzyme (Cat. No. 11766051) which is specific to dsDNA and heat-labile, hence does not require harsh inactivation conditions.

In comparing the different SuperScript III kit formats, I notice that some utilize a 10X buffer and others a 5X. The recipes are also slightly different - why is this?

It is recommended to use the buffer that comes supplied with the enzyme. The reasons for the slight differences are that the kits were developed at different times, possibly by different R&D groups.

Does SuperScript III exhibit TdT activity?

SuperScript III exhibits low TdT activity. If TdT activity is required, use our SuperScript IV RT or SuperScript IV Template Switching RT Master Mix.

What is the difference between SuperScript III RT and the RT in the SuperScript VILO kit?

The SuperScript VILO cDNA Synthesis Kit contains a mix of SuperScript III RT and helper proteins which help to increase the efficiency of the reverse transcription reaction and thus improve yield. The RT in the SuperScript VILO kit is active at 42 degrees C due to the helper proteins.

Does Platinum Taq DNA Polymerase High Fidelity enzyme mix leave 3' A-overhangs on the PCR product for subsequent cloning into a TOPO TA or original TA vector?

Yes, the enzyme mix leaves 3' A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3' A-overhangs to the product for TA cloning.

What is the difference between Platinum technology and AccuPrime technology?

With Platinum technology, anti-DNA polymerase antibodies bind to the enzyme until the denaturing step at 94 degrees C, when the antibodies degrade. The polymerase is now active and primer extension can occur. AccuPrime Taq combines Platinum Taq (Taq + Platinum antibodies) with proprietary thermostable AccuPrime accessory proteins. The 10X reaction buffer contains the accessory proteins which enhance specific primer-template hybridization during each cycle of PCR.

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