igScript™ Reverse Transcriptase (igScript RT) is a recombinant Moloney Murine Leukemia Virus (MMLV) reverse transcriptase with reduced RNase H activity. It is highly efficient at producing full-length cDNA from long RNA templates. igScript RT is an RNA-directed DNA polymerase which lacks 3´ → 5´ exonuclease activity and is capable of producing cDNA from as little as 50 pg of total RNA for real-time RT-PCR analysis and other applications.
The physical purity of this enzyme is ≥98% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).
The gene encoding recombinant MMLV Reverse Transcriptase with mutated RNase H domain is expressed in E. coli.
- First strand cDNA synthesis for PCR or RT-PCR
- Gene expression data validation by using RT-PCR product Includes
- Recombinant MMLV reverse transcriptase with greatly reduced RNase H activity.
- Active at temperatures up to 55°C.
- Highly efficient at producing full-length cDNA from as little as 50 pg of total RNA.
- igScript™ Reverse Transcriptase
- 10x igScript™ RT Reaction Buffer
1X MMLV Reverse Transcriptase Reaction Buffer
50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, pH 8.3 @ 25°C
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25ºC
One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid insoluble material in 10 minutes at 37 °C using poly r(A)/oligo (dT) as a substrate.
65°C for 20 minutes.
First Strand cDNA Synthesis Protocol
- In a sterile micro-centrifuge tube, add the following components on ice:
- Heat the reaction for 5 minutes at 65 °C. Spin down briefly. Place the tube immediately on ice.
- Add the following components:
- If using random hexamers, incubate the reaction at 25 °C for 10 minutes, then proceed to step 5.
- Incubate the reaction at 42 °C for 30-60 minutes.
- Inactivate the enzyme by incubating at 65 °C for 20 minutes.
- Store products at -20 °C or proceed to next step.
* RNase inhibitor can be purchased from any commercial source