Download HiScript ® Reverse Transcriptase

HiScript® Reverse Transcriptase
Catalog # R101
Introduction
HiScript® Reverse Transcriptase is a brand new reverse transcriptase based on mutagenesis of
M-MLV (RNase H-) Reverse Transcriptase. HiScript® Reverse Transcriptase is most active at
50°C, though highest reaction temperature could be set at 55°C, thus it is applicable to reverse
transcription of RNA templates with secondary structures. Elevated affinity of HiScript® Reverse
Transcriptase to the template makes reverse transcription reaction more efficient, with more
full-length cDNA can be obtained and as low as 1 pg total RNA can be detected, which is
especially applicable to a small amount of templates and the reverse transcription of low-copy
genes. In addition, the error rate of HiScript™ Reverse Transcriptase is lower than M-MLV
(RNase H-) Reverse Transcriptase, thereby increasing the fidelity of cDNA cloning.
Package Information
Components
5× HiScript® Buffer
HiScript® Reverse Transcriptase (200U/ μl)
R101-01 2,000U
500 μl
10 μl
R101-02 10,000U
500 μl
50 μl
Storage
Store at-20°C
Unit Definition
One activity unit (U) is defined as the amount of enzyme that will incorporate 10 nmol of dNTP
into acid-insoluble material in 10 minutes at 37°C, using Poly (rA)•Oligo (dT) as template/ primer.
Quality Control
Exonuclease activity: Incubation of 200 U for 1 hour at 37°C with 0.6 μg λ-Hind III does not
alternate its bands on agarose gel.
Endonuclease activity: Incubation of 200 U for 1 hour at 37°C with 0.6μg supercoiled pBR322
DNA does not alternate its bands on agarose gel.
Contamination of RNase: Incubation of 200 U for 1 hour at 37°C with 1μg total RNA of mouse
liver does not alternate its bands on agarose gel.
Functional test
1. Add 200U into the RT reaction and react for 45 min at 50°C with 500 ng Hela cell total RNA
as template and Oligo(dT)18 as primer. Take 1/ 10 of cDNA products to carry out PCR
amplification of DNCH gene. After agarose gel electrophoresis and EB staining, a clear 5.6
kb band can be detected.
2. Add 200U into the RT reaction, and react for 30 min at 50°C with 500 ng Hela cell total RNA
as template and Oligo (dT)18 as primer. Take 1/ 10 of cDNA products to carry out PCR
amplification ofβ-actin gene. After agarose gel electrophoresis and EB staining, a clear 550
bp band can be detected.
Protocol
1. Guidelines for PCR reaction
a. Denaturation of RNA template*
Set up the following mixture in RNase free centrifuge tube
RNase free ddH2O
to 13 μl
Oligo(dT)23 VN (50 μM)
1 μl
or Random hexamers (50 ng/μl)
1 μl
or Gene Specific Primers (2 μM)
1 μl
Total RNA
10 pg- 5 μg
Or Poly (A)+ RNA
10 pg- 500 ng
Heat reaction to 65 ℃ for 5 min, and quickly chill on ice for 2 min.
*RNA template denaturation help open the secondary structure can improve the yield of
first strand cDNA to a large extent. For longer than 3 kb cDNA fragment, do not omit the
denaturation step.
b. Preparation of a first strand cDNA synthesis reaction
Mixture of previous step
13 μl
5×HiScript® II buffer
4 μl
dNTP (10 mM each)
1 μl
HiScript® II reverse transcriptase (200U/ul)
1 μl
RNase inhibitor (40U/μl)
1 μl
Mix gently with a pipette.
c.
Under the following conditions of the first strand cDNA synthesis reaction
25°C1
10 min
42°C2
30 min
85°C
5 min
1. This step is required only when using the Random hexamers; using Oligo dT23VN or Gene Specific
Primer omits this step.
2. If the template has a complicated secondary structure, the reaction temperature was raised to 55°C, helps
to increase production
The product can be used in PCR reactions immediately, or store at -20°C, and used within
six months; after long-term storage is recommended aliquots at -80°C. cDNA Avoid
repeated freezing and thawing.
2.
a.
Guidelines for qPCR reaction
Preparation of a first strand cDNA synthesis reaction mixture
Set up the following mixture in RNase free centrifuge tube
RNase free ddH2O
to 20 μl
5× HiScript® II Buffer
4 μl
dNTP (10 mM each)
1 μl
HiScript® II Reverse Transcriptase (200U/ μl)
1 μl
RNase inhibitor (40 U/ μl)
1 μl
Oligo(dT)18 (50 μM)
1 μl
or Random hexamers (50 ng/ μl)
1 μl
Total RNA
10 pg- 1 μg
or Poly A+ RNA
10 pg- 100 ng
b. Under the following conditions of the first strand cDNA synthesis reaction.
25°C
50°C*
85°C
5 min
15 min
5 min
* For templates with complex secondary structure, raise temperature to 55℃ to improve cDNA increment.
The product can be used in PCR reactions immediately, or at -20°C, and used within six
months; after long-term storage is recommended aliquots at -80°C. cDNA Avoid repeated
freezing and thawing.