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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.
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
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.
First - Strand Synthes is System for RT - PCR 50 Preps (RT_0212-02.pdf, 180 Kb) [Download]
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