RTase, Recombinant Reverse transcriptase, RNase H

Description

Description

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.

 Application

• 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

Component	Volume
Total RNA, or miRNA	50 ng-5 μg
HiPureAnchored-Oligo (dT)18 primer (0.5 μg/μl)	1 µl
HiPure-Random hexamer primer (0.1 μg/μl) Or Gene Specific primer	1 µl   2 pmol
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

Component	Volume
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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