TRANSCRIPTME is a modified, recombinant form of the Reverse Transcriptase from Moloney Murine Leukemia Virus (M-MuLV) purified from Escherichia coli. TRANSCRIPTME Reverse Transcriptase synthesizes a complementary DNA strand in the presence of a primer using either RNA (cDNA synthesis) or single-stranded DNA (ssDNA) as a template.
Available Options:
| SKU | Unit | ||
|---|---|---|---|
 | RT32-010 | 10 000U (200 U/µl) | Ask for an offer |
| RT32-050 | 50 000U (200 U/µl) | Ask for an offer |
TRANSCRIPTME is a modified, recombinant form of the Reverse Transcriptase from the Moloney Murine Leukemia Virus (M-MuLV) purified from Escherichia coli. TRANSCRIPTME synthesizes a complementary DNA strand in the presence of a primer using either RNA (cDNA synthesis) or single-stranded DNA (ssDNA) as a template.
TRANSCRIPTME Reverse Transcriptase has increased thermal stability, which allows the reaction to be carried out at a higher temperature (optimum activity at 50°C). It increases the efficiency and specificity of those transcribed RNA regions which are rich in GC pairs and/or contain secondary structures. The enzyme has no 3’ –› 5’ exonuclease or RNase H activity, which improves the synthesis of a full-length cDNA, even from long mRNA templates, using random priming. The enzyme gives high yields of first strand cDNA synthesis up to 7 kb long.
Features and advantages
- High yields of full-length cDNA synthesis (up to 7 kb long)
- RNA and DNA-dependent DNA polymerase activities
- Increased sensitivity in RT-qPCR and RT-PCR assays
- Starting material: 10 pg – 5 µg of total RNA or 10 pg – 500 ng of mRNA
- Optimal reaction temperature: 50°C
- Increased thermostability
- No RNase H or 3’–› 5’ exonuclease activities
- Suitable for the amplification of difficult RNA templates
Applications
- Full-length cDNA template synthesis for RT-qPCR and two-step RT-PCR assays
- cDNA synthesis for molecular cloning
- cDNA library construction
- RNA analysis
10x RT Reaction Buffer
500 Tris-HCl (pH 8.3), 750 mM KCl, 30 mM MgCl2, 100 mM DTT
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TRANSCRIPTME Reverse Transcriptase (RT32) – Troubleshooting
Additional information
- We recommend the EXTRACTME TOTAL RNA KIT (EM09.2) and EXTRACTME TOTAL RNA PLUS KIT (EM11.2) for total RNA isolation from tissues and cell cultures
- During RT-PCR reaction mixture preparation, keep the TRANSCRIPTME Reverse Transcriptase and 10x RT Reaction Buffer on ice or in a freezing rack
- Use an RNase H treatment for reactions sensitive to residue RNA traces in order to increase the sensitivity of the RT-qPCR
- The quantity of cDNA used when preparing PCR or qPCR reactions should not exceed 1/10 of the final reaction volume; e.g. a maximum volume of 2.5 µl of cDNA should be used in a 25 µl reaction
- The activity of TRANSCRIPTME Reverse Transcriptaseis inhibited by metal ion chelating agents (e.g. EDTA), inorganic phosphor, pyrophosphate and polyamines
- Enzyme inactivation should be carried out at 85°C for 5 min.
Working with RNA
Acquisition of high quality, intact RNA, free of genomic DNA and RNase traces, is vital for the synthesis of a full-length cDNA followed by an accurate quantitative analysis (qPCR). The following recommendations for working with RNA should therefore be followed:
- Maintain aseptic working conditions: use disposable gloves, changing them as frequently, as required; use RNase-free consumables; only work in an area assigned for working with RNA and with equipment designated for that purpose.
- DNase I enzyme (not included) may be used if obtaining a DNA-free RNA sample is required.
- RNA samples should be stored aliquoted at -70°C. Avoid subjecting the samples to repeated freezing and thawing cycles.
Quality control
The absence of DNase and RNase activities has been confirmed using the relevant procedures.
TRANSCRIPTME Reverse Transcriptase is > 90% pure as judged by SDS polyacrylamide gel. In addition, the functional quality is tested by RT-PCR experiment.
TRANSCRIPTME Reverse Transcriptase (RT32) – MSDS
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