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M-MLV Reverse Transcriptase (RNaseH-) 

M-MLV Reverse Transcriptase (RNaseH-) is a recombinant reverse transcriptase expressed and purified from E. Coil. This enzyme can be used in cDNA synthesis with long messenger RNA templates due to the lacking of RNase H activity by point mutation.
Cat. No. Size Price Quantity  
TB10004A 5000 U USD 69
TB10004B 20000 U USD 220
Tel: +1 (973) 789-8318Email: [email protected]

Reagents Supplied

  • M-MLV Reverse Transcriptase (RNaseH-)200U/μl
  • 5× M-MLV Reaction Buffer

Description

M-MLV Reverse Transcriptase (RNaseH-) is a recombinant reverse transcriptase expressed and purified from E. Coli. This enzyme can be used in cDNA synthesis with long messenger RNA templates due to the lacking of RNase H activity by point mutation.

cDNA Synthesis

1.Add the following components to a nuclease-free microcentrifuge tube for a 20μl.

RNA 0.01-5 μg total RNA or 1 pg-0.5 μg mRNA
OligodT or Random primer 1 μl
RNase-free H2O up to12.5 μl

2. Incubate at 65°C for 5 min and quick chill on ice,spin briefly to collect the solution and add:

5 × M-MLV Reaction Buffer 4 μl
RNase Inhibitor 0.5 μl
dNTP mix(2.5 mM each) 2 μl
M-MLV Reverse Transcriptase (RNaseH-)(200U/μL) 1 μl

3.Mix gently, and incubate for 60 minutes* at 42°C for OligodT or at 25°C for 5 minutes prior to 42°C for 60 minutes* for random primer.

4.Inactivate the reaction by heating at 70°C for 5 minutes.

*Incubation time can vary from 30 min to 60 min. Prolonging reaction time may help to obtain longer cDNA

Notes

1. All procedure are performed on ice.

2. cDNA can be stored at -20°C or be used as a template for amplification directly.

3.Removing RNA complementary to the cDNA may be needed for amplification of some PCR targets(>1 kb), add 1 μl (2 units) of RNase H and incubate at 37°C for 20 minutes to remove RNA.

Quality Control

Tested for contaminating endonucleases, exonucleases, and ribonucleases by SDS-PAGE.

Prepare PCR Reaction

Use 10% or less of the first-strand reactionfor PCR. Normally 2 μl of the reaction from “Synthesize first-strand cDNA using M-MLV RT” used for 50 μl PCR reaction.

A suggested PCR reaction1

cDNA 1~2 μl
Forward primer(10 μM) 1 μl
Reverse primer(10 μM) 1 μl
2×PCR Master Mix 25 μl
ddH2O up to 50 μl

1: PCR reaction set-up should follow the PCR polymerase manual.

Troubleshooting

1:No or faint bands after analysis of amplified products.

◆Degraded RNA: Your RNA might have been degraded after the extraction. Check the integrity of your RNA with an agarose gel run.

◆Low purity of RNA: Wash of the mRNA pellet carefully with 70% (v/v) ethanol.

◆Wrong primer: Switch from oligo(dT) to a random primer or you might benefit from a mixture of the two kinds of primers.

◆Secondary structure on the RNA: Increase the cDNA synthesis temperature to 55°C.

2.Amplified product larger than expected

◆Contamination by genomic DNA: pretreat your sample with DNase I

3.Amplified product in negative control

◆Contamination by genomic DNA: pretreat your sample with DNase I

Datasheet Download

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