Reverse Transcriptase, Avian Myeloblastosis Virus (RT)

Source: \nAvian myeloblastosis virus particles\n\nStorage Buffer: \n200mM potassium phosphate (pH 7.2), 0.2% Triton X-100, 2mM DTT and 50% glycerol\n\nReaction Buffer (5X): \n250mM Tris-HCl (pH 8.3 @ 25 degrees C), 250mM KCl, 50mM MgCl2, 2.5mM spermidine and 50mM DTT.\n\nUnit Definition: \nOne unit is the amount of enzyme required to catalyze the transfer of 1nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37 degrees C. Reaction conditions are: 50mM Tris-HCl (pH 8.3), 8.75mM MgCl2, 40mM KCl, 10mM DTT, 0.1mg/ml BSA, 1mM radiolabeled dTTP and 0.25mM poly(A)400 and 0.25mM oligo(dT)50.\n\nQuality Control: \nFirst-Stand cDNA Synthesis: First strand cDNAs, of 1.2 kb Control RNA is synthesized using 30 units of enzyme, 1ug of each template, an oligo(dT) primer and a radiolabeled dNTP. The minimum specification is the production of 120ng of first-strand cDNA. Full-length cDNA must be observed by gel electrophoresis and autoradiography.\n\nEndonuclease Activity:\n1ug of Type I supercoiled plasmid DNA is incubated with 25 units of enzyme in 50 mM Tris (pH 8.3), 40 mM KCl, 7mM MgCl2, 10 mM DTT for one hour at 37 degrees C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting.\n\nNuclease Activity: \n50ng of radiolabeled DNA or RNA is incubated with 25 units of enzyme in 4mM Tris (pH 8.3), 3.2mM KCl, 0.56mM MgCl2, 0.8mM DTT for one hour at 37 degrees C, and the release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Passing specifications is < 1% release for DNase and < 3% release for RNase.\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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