RTase, Recombinant Reverse transcriptase enzyme is an engineered version of M-MLV reverse transcriptase with deficient RNase H activity. The enzyme is purified from E. coli expressing a portion of the gene of M-MLV on a plasmid. The enzyme is used to synthesize first-strand cDNA up to 8 kb.
- cDNA Synthesize Protocol
- A 20-μL reaction volume can be used for 50 ng–5 μg of total RNA.
- Add the following components to a nucleasefree microcentrifuge tube, Table1.
Table1: First step in cDNA synthesis
| ||50 ng-5 μg|
| ||1 µl|
|1 µl |
|RT-PCR Grade Water||To 12 µl|
1. Heat mixture to 65°C for 5 minutes 2. Quick chill on ice for 2 min. 3. Collect the contents of the tube by brief centrifugation and add the ingredient as written in table2. Table2: Second step in cDNA synthesis
|5 × RT-Buffer (specialized for RTase)||4 µl|
|HunteRNase, Recombinant (50 U/μl) *||1 µl|
|RTase, Recombinant Reverse transcriptase||1 µl|
|HiPure-dNTP mix (10 mM, 2.5 mM each)||2 µl|
4. For HiPure-Anchored-Oligo (dT)18 primer or gene specific primer, incubate at 42 ̊C for 30-60 min. Note: In case of using HiPureRandom hexamer primer (0.1 μg/μl), incubate at 25 ̊C for 10 min, then at 42 ̊C for 30-60 min. 5. Incubate at 85 ̊C for 15 s to inactive enzymes. The cDNA can now be used as a template for amplification in PCR. However, amplification of some PCR targets (>1 kb) may require the removal of RNA by RNase treatment
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