Description:
Super M-MuLV Reverse Transcriptase (RT) is a genetically modified M-MuLV RT. It differs from the M-MuLV RT by its structure and catalytic properties. Full-length copies of large mRNAs, >10kb, may be synthesized. Super M-MLV Reverse Transcriptase has a much lower RNase H activity than AMV Reverse Transcriptase resulting in high yields of full length cDNA. This makes M-MLV Reverse Transcriptase very useful in cDNA synthesis and RT-PCR.
Source:
E. Coli cells with a cloned fragment of the pol gene encoding Moloney Murine Leukemia Virus reverse transcriptase.
Note:
M-MuLV RT has much lower RNase H activity than Avian Myeloblastosis Virus (AMV) reverse transcriptase.
Features:
Efficient synthesis of first strand cDNA up to 13 kb.
Incorporates modified nucleotides (e. G. Cy3-, Cy5-, rhodamine-, aminoallyl-, fluorescein-labeled nucleotides).
Inhibition and Inactivation:
Inhibitors: Metal chelators, inorganic phosphate,
Pyrophosphate and polyamines.
Inactivated by heating at 70º C for 10min.
Applications:
RT-PCR and real-time RT-PCR.
Synthesis of cDNA for PCR, cloning, and hybridization probes.
Generation of labeled cDNA probes for micro arrays.
First strand cDNA synthesis.
DNA labeling.
Analysis of RNA by primer extension.
Unit Definition:
One unit is that amount of enzyme required to incorporate 1 nmol of deoxynucleotide into DE-81 filter-binding material in 10 min at 37º C using poly (rA)-oligo(dT)12 - 18 as template-primer
Assay Conditions:
The reaction mixture (25 μ L) contains 50mM Tris-HCI (pH 8.3), 40mM KCl, 6mM MgCl2, 1mM DTT, 400μ M poly (rA)-oligo(dT)12 - 18, 500μ M radiolabeled TTP and 0.1 - 0.5 units enzyme. Incubation is at 37º C for 10 min.
Storage Buffer:
20mM Tris-HCl (pH 7.5), 0.1M NaCl, 0.1mM EDTA, 5mM DTT, 0.1% Triton X-100 and 50% glycerol.
5X Reaction Buffer:
250mM Tris-HCl (pH 8.3 at 25º C), 250mM KCl, 20mM MgCl2, 50mM DTT.
