TRANSCRIPTME LYO Reverse Transcriptase (RT32L)

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, 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 and reduced 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.
RT32L, RT32L-100

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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

Applications

  • 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.

Storage conditions

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 conditions

Shipping in range of temperatures from dry ice to room temperature.

TRANSCRIPTME LYO Reverse Transcriptase (RT32L) - Manual

TRANSCRIPTME Reverse Transcriptase (RT32) – Troubleshooting

Additional information

  • 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.

Quality control

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.

TRANSCRIPTME LYO Reverse Transcriptase (RT32L) - SDS


TRANSCRIPTME LYO Reverse Transcriptase (RT32L) - CoA

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