The SuperScript® III 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–5 µg of total RNA. SuperScript® III 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 SuperScript® III RT is not significantly inhibited by ribosomal and transfer RNA, it may be used to synthesize first-strand cDNA from a total RNA preparation.
Using the SuperScript® III First-Strand System 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. For the PCR reaction, we recommend one of the following DNA polymerases: Platinum® Taq DNA Polymerase provides automatic hot-start conditions for increased specificity up to 4 kb, Platinum® Taq DNA Polymerase High Fidelity provides increased yield and high fidelity for targets up to 15 kb, and Platinum® Pfx DNA Polymerase provides maximum fidelity for targets up to 12 kb.
For Research Use Only. Not for use in diagnostic procedures.
The SuperScript™ III First-Strand Synthesis System for RT-PCR is supplied with the following: • Oligo(dT)20 (50 µM), 50 µl • Random hexamers (50 ng/µl), 250 µl • 10X RT buffer, 1 mL • 0.1 M DTT, 250 µl • 25 mM magnesium chloride, 500 µl • 10 mM dNTP mix, 250 µl • SuperScript III RT (200 U/µl), 50 µl • RNaseOUT (40 U/µl), 100 µl • E. coli RNase H (2 U/µl), 50 µl • DEPC-treated water, 1.2 mL • Total HeLa RNA (10 ng/µl), 20 µl • Sense Control Primer (10 µM), 25 µl • Antisense Control Primer (10 µM), 25 µl
Sufficient for 50 reactions, based on a 20 µl reaction size. Store at -20°C.