Alator - Model M101 -AMV Reverse Transcriptase

Size: Bulk. Concentration: 10 U/µL. Storage Buffer: 200mM potassium phosphate (pH7.2), 0.2% Triton X-100, 2mM DTT and 50% glycerol.

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Reaction Buffer 5X: 250mM Tris-HCl (pH8.3 @ 25° Q, 250mM KG, 50mM MgCh, 2.5mM spermidine and 50mM DTT.
Unit Definition: One unit is the amount of enzyme required to catalyze the transfer of lnmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37° C. Reaction conditions are: 50mM Tris-HCl (pH 8.3), 8.75mM MgCh, 40mM KC1, lOmM DTT, 0.1 mg/ml BSA, ImM radiolabeled dTTP and 0.25mM poly(A):oligo(dT).
Quality Control:

First-Stand cDNA Synthesis: First strand cDNAs, of 1.2kb Control RNA is synthesized using 30 units of enzyme, 1 ug of each template, an oligo(dT) primer and a radiolabeled dNTP. The specification is the conversion of >12% of mRNAto cDNA. Full-length cDNAmust be observed by gel electrophoresis and autoradiography.
Endonuclease Activity: 1 ug of Type I supercoiled plasmid DNA is incubated with 25 units of enzyme in 50mM Tris (pH8.3), 40mM KC1, 7mM MgCh, lOmM DTT for one hour at 37<>C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting.
Nuclease Activity: 50ng of radiolabeled DNA or RNA is incubated with 25 units of enzyme in 4mM Tris (pH8.3), 3.2mM KC1, 0.56mM MgCh, 0.8mM DTT for one hour at 37°C, and the release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Passing specifications is

Physical Purity: Purity is >80% in 2 bands [2 subunits) as judged by SDS-polyacrylamide gels with Coomassie blue staining.
Expiration: Twelve months from shipment date
Storage and Handling: -20°C

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