SuperScript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase, 100 rxns - 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.

Find additional tips, troubleshooting help, and resources within ourReverse Transcription and RACE Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

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

The SuperScript VILO cDNA Synthesis Kit (Cat. No. 11754050) 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.