igScript™ Glycerol-free Reverse Transcriptase 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.

Protein Purity:

The physical purity of this enzyme is ≥98% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).

Product Source

The gene encoding recombinant MMLV Reverse Transcriptase with mutated RNase H domain is expressed in E. coli.

Applications

• First strand cDNA synthesis for PCR or RT-PCR
• Gene expression data validation by using RT-PCR

Benefits

• 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.

Product Includes

1. igScript™ Reverse Transcriptase
2. 10x igScript™ RT Reaction Buffer

Storage Temperature
–20 °C

1X MMLV Reverse Transcriptase Reaction Buffer
50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, pH 8.3 @ 25°C

Storage Buffer
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25ºC

Unit Definition
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.

Heat Inactivation
65°C for 20 minutes.

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

First Strand cDNA Synthesis Protocol

1. In a sterile micro-centrifuge tube, add the following components on ice: reverse-transcriptase-pic2.

2. Heat the reaction for 5 minutes at 65 °C. Spin down briefly.

3. Place the tube immediately on ice. Add the following components: reverse-transcriptase-pic3.

4. 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.

6. Inactivate the enzyme by incubating at 65 °C for 20 minutes.

7. Store products at -20 °C or proceed to the next step.

* RNase inhibitor can be purchased from any commercial source

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

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