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First strand cDNA synthesis with Super Script II

Contributed by Dr.A.Gratchev

This method of first strand cDNA synthesis is more efficient than Ready-to-Go beads. You can use as little as 100ng total RNA and still detect you gene expression in PCR.

  1. Bring 2 µg total RNA in the volume of 20 µl with RNase free water
  2. Add 4 µl 10x DNase buffer (Ambion)
  3. Add 15 µl RNase free water
  4. Add 1 µl (2 U) DNase I (Ambion)
  5. Incubate 30 min at 37°C
  6. Inactivate DNase I for 10 min at 65°C
  7. Add 5 µl Glycogen (MBI Fermentas)
  8. Add 35 µl Isopropanol
  9. Incubate 30 min -20°C
  10. Centrifuge 20 min 14000 rpm at 4°C
  11. Aspirate supernatant
  12. Wash with 500 µl RNase free 70% EtOH
  13. Aspirate supernatant
  14. Dissolve in 10 µl RNase free water
  15. Add 1 µl (0.5 µg) Oligo dT primer (Invitrogen)
  16. Incubate 10 min at 70°C
  17. Transfer on ice
  18. Add 4 µl 5x first strand buffer (Invitrogen)
  19. 2 µl 0.1 M DTT (Invitrogen)
  20. 1 µl (40 U) RNase out (Invitrogen)
  21. 1 µl 10 mM (2.5mM each) dNTP (Invitrogen)
  22. Mix, preincubate 2 min at 42°C
  23. Add 0.5µl (100U) SuperScript II (Invitrogen)
  24. Incubate 50 min at 42°C
  25. Freeze at -20°C

Make 1:10 and 1:100 dilutions of your cDNA and test the DNase digestion and synthesis efficiency with PDH RT-PCR.

\METHODS\cDNA\super script II