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First Strand cDNA synthesis kit, 20 Preps, Biomiga


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Biomiga
FS01-01
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First-Strand Synthesis System for RT-PCR, 20 Preps, Biomiga

The First-Strand Synthesis System for RT-PCR is optimized to synthesize first-strand cDNA from purified poly(A)+ or total RNA. RNA targets from 100 bp to >12 kb can be detected with this system. The amount of starting material can vary from 1 pg to 5 μg of total RNA. Reverse Transcriptase is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme is used to synthesize cDNA at a temperature range of 42-55°C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. Because Synthesis System for RT is not significantly inhibited by ribosomal and transfer RNA, it may be used to synthesize first-strand cDNA from a total RNA preparation.

cDNA synthesis is performed in the first step using either total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers, or a gene-specific primer. In the second step, PCR is performed in a separate tube using primers specific for the gene of interest.

 

Kit Contents

Contents

Volume

Oligo(dT)20 (50 μM)

20 μl

Random hexamers (50 ng/μl)

100 μl

10× RT buffer*

400 μl

25 mM MgCl2

200 μl

0.1 M DTT

100 μl

10 mM dNTP mix

100 μl

SuperScript. III RT (200 U/μl)

20 μl

RNaseOUT. (40 U/μl)

40 μl

E. coli RNase H (2 U/μl)

20 μl

DEPC-treated water

480 μl

Total HeLa RNA (10 ng/μl)

8 μl

Sense Control Primer (10 μM)

10 μl

Antisense Control Primer (10 μM)

10 μl

Storage and Stability

Store at -20°C,The First-Strand Synthesis System for RT-PCR is stable for 1 year when stored at -20°C.

Quality Control

A minimum of 25 ng of a 353-bp RT-PCR product was obtained from 100 pg of total HeLa RNA and human β-actin primers. A minimum of 25 ng of a 6.8-kb RT-PCR product was obtained from 500 ng of total HeLa RNA and human Pol ε primers.

Summary of Procedure

279693124322270_929

 

 

First - Strand Synthes is System for RT - PCR (RT_0212-01.pdf, 180 Kb) [Download]

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