SuperScript™ III Reverse Transcriptase, 2,000 Units - FAQs

Which components of the SuperScript III First Strand Synthesis System for RT-PCR are available for purchase separately?

The following components are available as stand-alone items:

- Superscript III Reverse Transcriptase (Cat. Nos. 18080093, 18080044, 18080085)
- Oligo (dT)20 Primer (Cat. No. 18418020)
- Random hexamers (Cat. No. 48190011)
- 10 mM dNTP Mix (Cat. Nos. 18427013, 18427088)
- RNAseOUT Recombinant Ribonuclease Inhibitor (Cat. No. 10777019)
- E. coli RNAse H (Cat. Nos. 18021014, 18021071)

I am interested in generating cDNA from total RNA. What is the difference between SuperScript III Reverse Transcriptase and SuperScript III First Strand Synthesis System for RT-PCR?

SuperScript III Reverse Transcriptase (Cat. Nos. 18080093, 18080044, 18080085) contains the stand-alone enzyme and a vial each of 5X first-strand buffer and 100 mM DTT.

SuperScript III First Strand Synthesis System for RT-PCR is a complete kit that provides the SuperScript III Reverse Transcriptase and all the other components required for synthesis of first-strand cDNA from total or poly(A)- RNA. It includes:
- Superscript III Reverse Transcriptase
- Oligo (dT)20 Primer
- Random hexamers
- 10X RT buffer
- 25 mM MgCl2
- 0.1 M DTT
- 10 mM dNTP Mix
- RNAseOUT Recombinant Ribonuclease Inhibitor
- E. coli RNAse H
- DEPC-treated water
- Total HeLa RNA control
- Sense control primer
- Anti-sense control primer
Note: The kit does not include the PCR amplification enzyme.

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.

How much of the first-strand cDNA reaction should I load for PCR?

While the volume is dependent on the starting amount of RNA used for the first-strand synthesis and the abundance of the target gene, we'd recommend starting with 10% of the first-strand reaction for your PCR reaction.

Why do I get low number of cDNA clones using SuperScript RT?

A low number of cDNA clones could result due to the reasons listed below:

(1) Phenol extractions were performed with phenol that was equilibrated with water that was not DEPC-treated.

(2) Poor first strand yield.

(3) No RNase H was added to second-strand reaction.

(4) Second-strand reaction was performed at a temperature greater than 16 degrees C.

(5) Dilution of first-strand reaction was made incorrectly: exact dilution is crucial, because the pH of the second-strand reaction differs from that of the first-strand reaction.

(6) The size fractionation column ran too quickly, the column was allowed to dry before fractions were loaded, or the column was not washed thoroughly to remove ethanol.

(7) Competent cells were of poor efficiency: verify efficiency of transformation with a control DNA.

(8) There was an insufficient amount of cDNA in the ligation mix.

(9) If cells were transformed by electroporation, there may have been too much salt in the electroporation mix, which kills cells.

Why are my first-strand cDNA products small (less than 2 kb on an alkaline agarose gel)?

Please see listed below a few reasons why you may be getting small first-strand cDNA products:

(1) Template was degraded by RNase contamination: maintain aseptic conditions.

(2) Secondary structure is present in the RNA:
-Heat the RNA to 70 degrees C for 10 min and quick-chill on ice to denature the RNA. Increase the temperature of the first strand reaction up to 50 degrees C. Denature the RNA by treatment with 20 mM methylmercuric hydroxide (see Krug et. al. (1987) Methods Enzymol 152:316).
-Try alternate reverse transcriptase such as SuperScript III polymerase and perform first-strand synthesis at higher temperature.

(3) Alkaline gel electrophoresis was performed incorrectly: use a 1% alkaline agarose gel.

(4) For random-primed first-strand cDNA, an excessive concentration of primers was used: use 50 ng random hexamers/1-5 µg total RNA.

(4) Incorrect fractions were taken at the column chromatography step.

SuperScript II RT is supplied as a 200 units/µL stock. We need to use 50 units for cDNA synthesis. Can we dilute the enzyme with 5X RT buffer to make it 50 units/µL?

You can dilute SuperScript II RT in 1X first-strand buffer if you plan to use it immediately. The enzyme is not stable in that buffer for extended periods. You can also dilute the enzyme in the storage buffer (20 mM Tris HCl pH 7.5, 1 mM DTT, 0.01% NP40, 0.1 mM EDTA, 0.1 M NaCl 50% glycerol) and store it at 20 degrees C.

Also consider using SuperScript IV reverse transcriptase. This enzyme does not need to be diluted in order to perform optimally with a smaller amount of starting template.

Can I use a DNA-RNA hybrid as a template for M-MLV Reverse Transcriptase (Cat. No. 28025013, 28025021)? Can other reverse transcriptases, such as SuperScript reverse transcriptase, be used in the same way?

Yes, you can use a DNA-RNA hybrid as a template for M-MLV Reverse Transcriptase.

We have not tested this for SuperScript reverse transcriptases, so we cannot guarantee it would also work with those products.

This article can be used as a reference for additional information.

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

How long can I store the cDNA generated from my reverse transcription (RT) step?

You can store the cDNA at -20 degrees C for up to 1 week. For long-term storage, we recommend storing the cDNA at -70 degrees C.