MMLV reverse transcriptase

MMLV RT

Reagent Sharers

Alex Brown

Summary

This is a RT derived from Moloney Murine Leukemia Virus containing D200N, L603W, T330P, L139P and E607K, for high processivity and thermostability (Baranauskas, 2012 PMID: 22691702)

Expression/Production Information

Alex has posted the expression/purification protocol at protocols.io

How to get the plasmid/material/reagent

Currently in Alex´s hands, OBL, FreeGenes, iBio_Chile and UBA_Argentina
This is vector received from Alex and being used in Chile.

FreeGenes is also distributing this vector.

Terms

Under openMTA from Alex, being able to redistribute under openMTA from iBio/Chile too.

References

Baranauskas, 2012 PMID: 22691702

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Right now working with MMLV, everything has worked great!

We are optimizing the conditions of use, but it seems that it is an enzyme with very robust activity in different conditions.

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We are also working on the expression of MMLV in Buenos Aires. Everything goes fine so far, but we have a little doubt about the protocol you shared. The lysozyme concentration on Buffer B is 1mg/ml. Isn’t high? Have someone tried without lysozyme or using a lower concentration?

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Cesar Ramirez (in Chile) said they used 0.1 mg/ml and works.

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Another purification protocol for MMLV has been added to the collection in protocols.io
https://www.protocols.io/view/recombinant-expression-and-purification-of-codon-o-bhcvj2w6
It is working for us in Chile.
Thanks to Cesar Ramirez group!

Hey guys!
I think I saw some aggregration on my SDS-PAGE (didnt confirm by Western Blot, though).
This publication: https://pubmed.ncbi.nlm.nih.gov/19330487/ states, that MMLV tends to aggregate in Imidazol and suggests an on column-cleavage elution with high recovery rate.
I’ve also read (dont know where right now ^^), that MMLV needs to be cleaved, otherwise there is a huge decrease in acitivity.
Any thoughts on that? I will try on-column cleavage the next run I guess.

In case of any interest, we are using MMLV and Pfu Ss07d in One-Step PCR reactions. Traditionally we have used MMLV for cDNA synthesis at 50 ° C, but recently we tested at 65 ° C and it works almost the same. I attach a diagram that summarizes the conditions used.

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