Catalyzes the reverse transcriptation of RNA to cDNA at elevated temperatures in the presence of MnCl2
Suitable for high temperature synthesis of DNA. Synthesizes cDNA from RNA template. Results in greater specificity of primer hybridization and extension of RNA. Can reverse transcribe at elevated temperatures. Minimizes problems with strong secondary structure of RNA. Used for efficient PCR of DNA, containing problematic secondary structures. Applicable to RT-PCR; the same enzyme is used for both reverse transcription and following amplification of obtained cDNA template. Resistant to amplification inhibitors present in template DNA isolated from problematic samples.
40 mM Tris-HCl (pH 8.5) 1 mM MnCl210 mM dithiothreitol1 mg/ml bovine serum albumin0.4mM polyA(dT)0.5 mM (α-32P)TTP @ 10 μCi/ml Incubation is for 10 minutes at 50 °C in a total reaction volume of 50 μl
Incubation at 50 °C
500 Units;100 Units
50 mM Tris-HCl (pH 7.5) 0.1 mM EDTA5.0 mM dithiothreitol50% (v/v) glycerol Stabilizers
One unit is the amount of enzyme required to incorporate 1.0 nmole TTP into acid insoluble material at 50 °C in 10 minutes.