TRANSCRIPTME LYO Reverse Transcriptase is a freeze-dried form of a modified Reverse Transcriptase originated from Moloney Murine Leukemia Virus (M-MuLV) expressed in Escherichia coli. TRANSCRIPTME LYO 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.
TRANSCRIPTME LYO Reverse Transcriptase has increased thermal stability, that 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 and reduced RNase H activity, that 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
- Increased stability (transport at ambient temperature)
- High yields of full-length cDNA synthesis (up to 7 kb long)
- RNA- and DNA-dependent DNA polymerase activity
- Increased sensitivity in RT-LAMP, 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 3’ –› 5’ exonuclease activity
- Reduced RNase H activity
- Suitable for amplification of difficult RNA templates
- Full-length cDNA template synthesis for RT-LAMP, RT-qPCR and two-step RT-PCR assays
- cDNA synthesis for molecular cloning
- cDNA library construction
- RNA analysis
Reverse Transcriptase preparation
Reconstitute the liophilizate of TRANSCRIPTME LYO Reverse Transcriptase RT32L-100 with 500 μl of RT Resuspension Buffer. Mix carefully by inverting the tube several times. Do not vortex. Wait about 3 minutes and then spin briefly.
Upon arrival, TRANSCRIPTME LYO Reverse Transcriptase and all components should be stored at -20°C . After resuspension, enzyme should be stored at -20°C in a freezer without a defrost cycle.
Shipping in range of temperatures from dry ice to room temperature.
TRANSCRIPTME LYO Reverse Transcriptase (RT32L) - Manual
TRANSCRIPTME Reverse Transcriptase (RT32) – Troubleshooting
- We recommend EXTRACTME TOTAL RNA KIT (EM09.2) and EXTRACTME VIRAL RNA KIT (EM39) for total RNA isolation.
- During preparation of RT-PCR, RT-qPCR or RT-LAMP keep TRANSCRIPTME LYO Reverse Transcriptase and 10 x 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 RT-qPCR.
- The quantity of cDNA used when preparing PCR or qPCR reactions should not exceed 1/10 of a 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 LYO Reverse Transcriptase is inhibited by metal ion chelating agents (e.g. EDTA), inorganic phosphors, pyrophosphates 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.
- Use RIBOPROTECT Hu RNase Inhibitor LYO (RT35L) or RIBOPROTECT Hu RNase Inhibitor (RT35) in upstream and downstream applications to avoid RNA degradation.
- HL-dsDNase (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.
The absence of DNase and RNase activities has been confirmed using the relevant procedures.
TRANSCRIPTME LYO Reverse Transcriptase is > 90% pure as judged by SDS polyacrylamide gel. In addition, the functional quality is tested by RT-PCR experiment.