Low Copy Number RNA Prep (for RT-PCR)

  1. Grow 5 ml of cells in YPD O/N.
  2. Dilute cells in fresh YPD, start growing from OD600 0.25
  3. Take 1 ml samples at OD ~0.6-0.8.
  4. Spin down at max RPM for 2 minutes.
  5. Discard supernatant (be careful not to disturb the pellet).
  6. Resuspend in 150 ul of TES (10 mM NaAc, 10 mM EDTA, 0.5 % SDS) and vortex.
  7. Add 150 ul of phenol and vortex, Incubate at 65˚C for 20 min.
  8. Quick freeze in liquid nitrogen. Remove tubes from liquid nitrogen, leave at RT for 3 minutes.
  9. Centrifuge at max RPM for 10 minutes at 4˚C.
  10. Transfer 100 ul of upper aqueous layer to fresh eppendorf tube.
  11. Add 100 ul of phenol/chloroform/IAA and vortex.
  12. Centrifuge at max RPM for 10 minutes at room temperature.
  13. Transfer 60 ul of upper aqueous layer to fresh eppendorf tube.
  14. Add 7.5 ul of 2.6M NaAc (or 6 ul of 3M NaAc), 200 ul of 100% ethanol, vortex.
  15. Incubate at –20 C for 30 minutes.
  16. Centrifuge at max RPM for 15 minutes at 4˚C. Carefully remove supernatant completely.
  17. Dry pellet in speed vacuum for 3 minutes.
  18. Resuspend pellet in 60 ul of RNase free H2O.
  19. Measure RNA concentration by dilute 10 ul RNA sample into 190 ul RNase free H2O. Before measuring, pre-warm spectrometer 5 minutes to let the ultra-violet lamp stable.
  20. Take 1 ug RNA sample for Dnase I treatment (Invitrogen Deoxyribonuclease I, Amplification Grade. Cat. No. 18068-015).

    RNA Sample x ul (1 ug)
    RNase free H2O 8-x ul
    10x Dnase I reaction Buffer 1 ul
    DNase I 1 ul
    10 ul

    Incubate tubes at RT for 15 minutes. Inactivate the DNase I by the addition of 1 ul of 25 mM EDTA solution to the reaction mixture. Heat for 10 minutes at 65 C.

  21. Store in freezer, but best results when used immediately.
  22. Use 1 ul of this RNA for standard RT reaction.