FicoScript®Reverse Transcriptase（反转录酶） is an engineered version of M-MLV RT with reduced RNase H activity and increased thermal stability. The enzyme is purified to near homogeneity from E. coli containing the modified pol gene of Moloney Murine Leukemia Virus . The enzyme can be used to synthesize first-strand cDNA at temperatures up to 48°C, providing increased specificity, higher yields of cDNA, and more fulllength product than other reverse transcriptases. It can generate cDNA from 100 bp to >12 kb.It is suitable for qPCR.
1 、 Enzyme Storage Buffer: M-MLV Reverse Transcriptase is supplied in 20mM Tris-HCl (pH 7.5), 200mM NaCl, 0.1mM
EDTA, 1mM DTT, 0.01% Nonidet P-40 and 50% glycerol.
2 、 M-MLV Reverse Transcriptase 5X Reaction Buffer: When the M-MLV Reverse Transcriptase 5X Reaction Buffer supplied with this enzyme is diluted 1:5, it has a composition of 50mM Tris-HCl (pH 8.3 25°C ), 75mM KCl, 3mM MgCl 2 and 10mM DTT.
3 、 Source: Purified from an E. coli strain expressing a recombinant clone.
4 、 Storage Conditions: Store at – 20°C . Avoid exposure to frequent temperature changes.
5 、 Unit Definition: One unit is defined as the amount of enzyme required to catalyze the transfer of 1nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37°C . The reaction conditions are: 50mM Tris-HCl (pH 8.3), 7mM MgCl 2 ,
40mM KCl, 10mM DTT, 0.1mg/ml BSA, 0.5mM [ 3 H]dTTP, 0.025mM oligo(dT) 50 , 0.25mM poly(A) 400 and 0.01% NP-40.
6 、 Usage Note: M-MLV Reverse Transcriptase is less processive than AMV Reverse Transcriptase, and therefore, more units of the M-MLV enzyme are required to generate the same amount of cDNA as in the AMV reaction. Thus, starting with 1μg of mRNA
in a first-strand cDNA synthesis, 200 units of the M-MLV enzyme are recommended as opposed to 25 units of the AMV enzyme.
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