Cat. No. : AM0670
Concentration : 200 Units/ μl
Volume : 50μl
Storage : -20 ℃ |
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| Description: |
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HiScript I Reverse Transcriptase is a multiple-point mutanted version of M-MLV RT. The enzyme is purified
from E. coli containing the mutanted pol. gene of Moloney
Murine Leukemia Virus. The enzyme can be used to synthesize
first-strand cDNA at higher temperatures than M-MLV RT,
providing increased specificity , higher yield of cDNA. |
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| Form: |
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20 mM Tris-HCl (pH 7.8)
100 mM NaCl
0.1 mM EDTA
1 mM DTT
50 % glycerol |
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| Components |
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Script RT
5X First-Strand Buffer
(250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2)
0.1 M DTT |
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| Unit Definition |
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One unit incorporates 1 nmole of dTTP into acid precipitable
material in 10 mins at 37℃ using poly(A)-oligo(dT) as template
primer . |
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| Standard protocol for First-Strand cDNA synthesis |
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- Add the following components to a microtube.
Oligo dT primer 50 pmole /Random primer 50 pmole
/ Gene specific primer 2 pmole
dNTPs Mixture (10 mM each)… 1 μl
Template RNA
( total RNA ≦ 5 μg or mRNA ≦ 1μg)
Sterile , distilled water to 12μl
- Heat at 65℃ for 5 mins, and cool immediately on ice.
Collect the contents of the tube by brief centrifugation.
- Prepare the reaction mixture by combining the following
reagents to a total volume 20μl .
Template RNA / Primer mixture … 12 μl
5X First-Strand Buffer …………… 4 μl
0.1 M DTT ……………………… 2 μl
RNase Inhibitor (optional)* ……… 1 μl
HiScript I Reverse Transcriptase … 1 μl
- Mix gently and spin down .
- Perform the reaction under the following condition
30℃ 10 mins* → 42 ( ~ 48 ) ℃** 30~60 mins.
- Heat at 70℃ for 15 mins.
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* This step is required for random primer.
** It is generally recommended to perform the RT reaction at
42℃ with this enzyme . However , if the reverse primer for
PCR is also used as a RT primer , non-specific products may be
amplified due to mispriming . In such a case , it is
recommended to perform the RT reaction at 48℃ . |
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| PCR |
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Use only 2μl of the First-Strand reaction for PCR . |
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- Add the following components to a PCR tube .
10X PCR Buffer……………… 5 μl
10 mM dNTPs Mixture………… 1 μl
10μM Forward primer ………… 1 μl
10μM Reverse primer …………1 μl
5U/μl Taq DNA polymerase … 0.4 μl
The First-Strand reactant…………2 μl
Autoclaved , distilled water to 50μl
- Mix gently and spin down .
- perform 20 to 40 cycles of PCR .
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