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RT-PCR M.12: RT PCR PROTOCOL, USING SUPERSCRIPT REVERSE TRANSCRIPTASE - (from Life Technologies Technical Product Information )                                                   Go Home

First Strand cDNA Synthesis: A 20 ul reaction volume can be used for 1-5 ug of total RNA or 50-500 ng of mRNA. Add the following components to a nuclease-free microcentrifuge tube:

Heat mixture to 70oC for 10 min and quick chill on ice. Collect the contents of the tube by brief centrifugation and add: 2. Mix contents of the tube gently and incubate at 42oC for 2 min.

3. Add 1 ul (200 units) of SUPERSCRIPT II, mix by pipetting gently up and down.

4. Incubation at 25oC for 10 min

5. Incubate 50 min at 42oC.
6. Inactivate the reaction by heating at 70oC for 15 min.

NOTE: The cDNA can now be used as a template for amplification in PCR. However, amplification of some PCR targets (those>1 kb) may require the removal of RNA complementary to the cDNA. To remove RNA complementary to the cDNA, add 1 ul (2 units) of E. coli RNase H and incubate 37oC for 20 min.

II. PCR Reaction
Use only 10% of the first strand reaction for PCR. Adding larger amounts of the first strand reaction may not increase amplification and may result in decreased amounts of PCR product.

1. Add the following to a PCR reaction tube for a final reaction volume of 100 ul:
2. Mix gently and layer 2 drops (~100 ul) of mineral oil over the reaction.
3. Heat reaction to 94oC for 3 min to denature.

4. Perform 15 to 40 cycles of PCR.