RTase, Recombinant Reverse transcriptase, RNase H

 12,090,000

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.

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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 

Technical Assistance

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Contact our technical support at any time by selecting one of these ways:

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Additional information

Size

10000 units

Specification

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Protocol

none!

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Resources

MSDS RTase, Recombinant

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