Applications:
- RT PCR
- Synthesis of cDNA
- mRNA 5’-end Mapping by Primer Extension Analysis
- End-labeling of DNA
- Dideoxynucleotide Sequencing

Benefit:

- Engineered M-MLV RT remains active up to 50°C increasing the
  length and yield of cDNA compared to WT version.

- Able to efficiently detect and produce cDNA in reactions that have
  less than 100 pg of RNA template.

- Amplification up to 8kb.

- reduced RNase H activity (RNase H–) than the wild type M-MLV RT.

Description: 
M-MuLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (M-MuLV RT) is an RNA-dependent DNA polymerase that synthesizes the cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. M-MuLV RT will also extend primers hybridized to single-stranded DNA. Second strand cDNA synthesis can be achieved from some mRNA templates without an additional DNA polymerase.  

Concentration: 200 u/µl

Storage Buffer:
20 mM Tris-HCl pH 7.4, 100 mM NaCl, 0,1mM EDTA, 50 % Gycerol, 0,1% IGEPAL, 1 mM DTT

Reaction Buffer complete 10X: 
500 mM Tris-HCl (pH 8.3 at 25°C); 30 mM MgCl₂; 750 mM KCl; 100 mM DTT.

Dilution Buffer 1X:

10 mM KH₂PO₄ (pH 7.5); 0,1 mM EDTA; 200 mM NaCl; 7 mM 2-mercaptoethanol; 50% glycerol

Unit definition:
One unit of the enzyme incorporates 1 nmol dTTP into acid-precipitable material in 10 minutes at 37°C, using poly(A) oligo dT as a template primer.

Transportation: on blue ice

Storage: at -20°C for 24 months

Notes: RT enzyme / MMLV RTase / RNA template / Complementary DNA / cDNA / Reverse Transcription / Molecular cloning / RNA sequencing / PCR / Genome Analysis / superscript

Usage:  
Standard Protocol: We recommend to prepare 2 Mixes

Mix I

Mix II

^1.) 30 min for cDNA with 500 bp; 115 min for 1,5 kb
^2.) depends on the RNA: Higher temperatures (up to 55 °C) for higher structured RNA; Try to adjust the pH to 8.8

Note: MMLV used under standard 37°C reaction conditions is best for synthesis of 7 kb or less.

Increasing reaction temp to 42°C may allow synthesis up to 10 kb. MMLV activity drops drastically above 42°C.

Quality control:
Endonuclease Activity:  1 µg of Type 1 supercoiled plasmid DNA is incubated with 500 units of enzyme in 1X reaction buffer for one hour at 37°C. The supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify absence of nicking or cutting. 

Nuclease Activity:  50 ng of radio labelled DNA or RNA is incubated with 200 units of enzyme in 1X reaction buffer for one hour at 37°C, resulting in <1% release for both DNase and RNase.

Purity:  >90% as judged by SDS-polyacrylamide gels with blue staining. MMLV RT is free of detectable RNase, and DNase (exo- and endonuclease) activities.