|TranscriptMe Reverse Transcriptase
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 hasincreased 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 up to 10 kb long.
Features and advantages
– High yields of full-length cDNA synthesis (up to 10 kb long)
– RNA- and DNA-dependent DNA polymerase activities
– Increased sensitivity in RT-qPCR and RT-PCR assays
– Starting material: 10 pg – 5 g 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
– Full-length cDNA template synthesis for RT-qPCR and two-step RT-PCR assays
– cDNA synthesis for molecular cloning
– cDNA library construction
– RNA analysis
10 mM Tris-HCl (pH 8.0), 80 mM NaCl, 0.2mM EDTA, 50% (v/v) glycerol
10x RT Reaction Buffer
500 Tris-HCl (pH 8.3), 750 mM KCl, 30 mM MgCl2, 100 mM DTT
- We recommend the EXTRACTME TOTAL RNA KIT (EM09) and EXTRACTME TOTAL RNA PLUS KIT (EM11) 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.
Store all components at -20°C. The stability of the reagents can be extended if stored at -70°C. The product is stable for 12 months providing it is stored and used correctly.
Shipping on dry ice