In this first step, we use a gene-specific primer of ALK to obtain a 5' extended cDNA. Work on wet ice while setting up the reaction and use sterile gloves and RNAse free materials. Supersript™ II RT is not inhibited significantly by ribosomal and transfer RNA, and may be used effectively to synthesize first strand cDNA from a total RNA preparation.
1. Add the following to a thin-walled PCR tube
DEPC-treated water to a final volume of 15.5 pE
2. Incubate the mixture 10 min at 70°C to denature RNA. Chill 1 min on ice. Collect the contents of the tube by brief centrifugation and add the following in the given order:
10X PCR buffer 2.5 (L
Final volume 8.5 (L
3. Mix gently and collect by brief centrifugation. Incubate for 1 min at 42°C.
4. Add 1 pL of Superscript™ II RT. Mix gently and incubate for 50 min at 42°C. (see Note 3).
5. Incubate at 70°C for 15 min to terminate the reaction.
6. Centrifuge 10-20 s and place reaction at 37°C.
7. Add 1 pL of RNAse mix, mix gently but thoroughly and incubate for 30 min at 37°C.
8. Collect the reaction by brief centrifugation and place on ice. The procedure may be stopped at this point.
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