Reverse transcriptase MMLV H-.
Description	Reverse transcriptase M-MLV RNaseH- (EC 2.7.7.49) is a modified Moloney murine leukemia virus reverse transcriptase lacking RNase H activity. The enzyme displays low processivity and elongates DNA chains slowly and tends to pausing and termination during polymerization; error rate is low (1 mismatched residue in 30.000 incorporated). In contrast to most other DNA polymerases, RT can use either RNA or DNA to prime DNA synthesis from an RNA or DNA template. M-MLV RNaseH- is much less sensitive than AMV RT to inhibition by tRNA and rRNA, and well suited for copying unfractionated mRNA.
Unit definition	1 unit of the enzyme incorporates 1 nmol of dTTP into acid-insoluble form in 10 min at 37°C using 250 µM poly(A)400 and 25 µM oligo(dT)50. The specific activity of M-MLV H- is 100.000U/mg protein.
Reaction buffer	B16 (10x): 250 mM Tris-HCl pH 8.3, 150 mM KCl, 40 mM MgCl2, 50 mM DTT.
Reaction conditions	High concentration of dNTPs (>0,5 mM) in a reaction mixture results in the most efficient synthesis of full-length cDNAs. Spermidine-HCl at 0.5 mM stimulates the activity of AMV and Rauscher murine leukemia virus RT and inhibits M-MLV H-. DTT at a final con. of 5-10 mM is necessary.
Storage	Store at -20°C. Freezing the enzyme does not affect the unit activity, but will decrease the functional ability to copy long mRNAs.
Applications	cDNA synthesis; production of strand-specific radiolabeled probes; filling in and labeling of DNA fragments with 5' protruding ends; blunt end formation during the second-strand reaction.
Quality control	activity, SDS-PAGE-purity, absence of endonuclease/nickase, RNase and DNase activities, specific performance tests
References	1. Tanese N., Goff, S.P. (1988) Proc. Natl. Acad. Sci. USA 85, 1977. 2. Roth, M. et al. (1985) J. Biol. Chem. 260, 9326.
Catalog #	L3.8	L3.9
Pack size	20.000 U	50.000 U