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View additional product information for SuperScript™ II Reverse Transcriptase - FAQs (18064071, 18064022, 18064014)
16 product FAQs found
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.
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.
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.
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.
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.
No, the DTT will need to be replaced.
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.
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.
We have not tested incorporation of biotin labeled nucleotides directly. Biotin should work as long as it is not substituted directly for its nucleotide replacement. Usually the biotin will be attached to one nucleotide such as TTP. We suggest that you try a reaction with a 1:1, 1:4, 1:10, and 1:50 replacement of the TTP in the reaction with biotin-labeled TTP. Most likely the 1:4 or 1:10 will work well. Having an excess of normal nucleotide around should space out the incorporation of biotin. This suggestion is speculative and has not been tested experimentally.
The following reagents will inhibit SuperScript II RT activity by at least 50%.
34% glycerol
4 µg/mL heparin
0.0025% (w/v) SDS
5% (v/v) formamide
17.0% (v/v) DMSO
4 mg/mL glycogen
30 mM guanidine-HCl
15 mM guanidine isothiocyanate
1 mM EDTA
2.5 mM NaPPi
0.4 mM Spermidine
The enzymes can be inactivated by adding a chelating agent such as EDTA. Alternatively, with the exception of ThermoScript RT and Thermo-X RT, the enzymes can be heat inactivated at 70 degrees C for 10 min.
ThermoScript RT should be heated to 85 degrees C for 5 min for complete inactivation.
For Thermo-X RT, if using an oligo(dT) primer, add EDTA to the reaction at a final concentration of 5 mM. Inactivate the reaction by heating at 90 degrees C for 5 min.
The optimal temperature for for MMLV is 42 degrees C. The optimal temperature for SuperScript II RT is 42 degrees C, and can be used up to 50 degrees C. The optimal temperature for SuperScript III RT is 50 degrees C, and can be used up to 55 degrees C. For some qRT-PCR reactions where gene-specific primers are used, you can do the RT reaction at 60 degrees C. The optimal temperature for SuperScript IV RT is 50 degrees C, but can be used up to 65 degrees C.
Include a control reaction where the RNA has not been incubated with reverse transcriptase to test for specificity. If this RNA gives a PCR product, it is most likely generated from genomic DNA contamination. Alternatively, a primer set spanning two different exons can be designed such that the PCR product from the cDNA would be of a different size compared to a product generated from genomic DNA. Primers may also be designed to span an exon/exon junctions. These primers are not likely to amplify from genomic DNA templates. For DNase treatment of RNA, we recommend using Amplification-Grade DNase I (Cat. No. 18068-015) or ezDNase Enzyme (Cat. No. 11766051).
It is not always necessary to digest the first-strand cDNA with RNase H. For many primer-template combinations, PCR products are seen without the RNase H treatment. Since SuperScript II and III RT lack RNase H and ThermoScript RT essentially is RNase H minus, the un-nicked RNA/cDNA hybrids may not denature well during the initial denaturation steps in PCR, leading to decreased sensitivity of the PCR reaction. These cDNA templates may require RNase H digestion. If a PCR product is not obtained when an RNase H step is not included after cDNA synthesis, always repeat the PCR after an RNase H treatment.
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.
SuperScript II Reverse Transcriptase and SuperScript First-Strand Synthesis System for RT-PCR are shipped on wet ice. Our packaging and gel ice are designed to last approximately 48 hours before rising above 8 degrees C. Overnight incubation under these shipping conditions does not affect the SuperScript II Reverse Transcriptase enzyme's activity, performance, or applicable expiry date.
Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.