M.12: RT PCR PROTOCOL, USING SUPERSCRIPT REVERSE TRANSCRIPTASE - (from Life Technologies Technical Product Information )
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:
- 1 ul 50-250 ng of random primers
- 1-5 ug Total RNA
- Sterile, distilled water to 12 ul
2. Mix contents of the tube gently and incubate at 42oC for 2 min.
- 4 ul 5X First Strand Buffer (250 mM Tris-Cl, pH 8.3,
- 375 mM KCl, 15 mM MgCl2). This buffer is supplied with the SuperScript Reverse Transcriptase.
- 2 ul 0.1 M DTT
- 1 ul 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH)
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:
- 10 ul 10X PCR Buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl]
- 3 ul 50 mM MgCl2
- 2 ul 10 mM dNTP Mix
- 2 ul Amplification Primer 1 (10 uM)
- 2 ul Amplification Primer 2 (10 uM)
- 1 ul Taq DNA polymerase (2-5 U/ul)
- 2 ul cDNA (from first strand reaction, preferably RNase H-treated)
- 80 ul Autoclaved, distilled water
- 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.