- Reverse transcriptase MMLV H-.
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- Description
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- 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.
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- Unit definition
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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.
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- Reaction buffer
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- B16 (10x): 250 mM Tris-HCl pH 8.3, 150 mM KCl, 40 mM MgCl2,
50 mM DTT.
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- Reaction conditions
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- 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.
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- Storage
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- Store at -20°C. Freezing the enzyme does
not affect the unit activity, but will decrease the functional ability
to copy long mRNAs.
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- Applications
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- 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.
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Quality control
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activity, SDS-PAGE-purity, absence of endonuclease/nickase, RNase and
DNase activities, specific performance tests
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References
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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.
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- Catalog #
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- L3.8
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L3.9
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Pack size
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20.000 U
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50.000 U
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