This is a cached page for the URL (http://www.pmci.unimelb.edu.au/core_facilities/manual/mb120.asp). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
RESEARCH DIVISION Laboratory Manual

 


 

Total RNA preparation

  1. Homogenise sample in minimum volume of TRIzol reagent to facilitate homogenisation,
    add TRIzol reagent to total 1mL, 5 mins RT
  2. Add 200無 chloroform, shake vigorously for 15 secs, 3 mins RT
  3. Spin 12000g at 4 degrees for 15 mins:
    Phases separate into three: bottom red, phenol chloroform; middle interphase; upper clear colourless aqueous phase (ca. 60% vol) containing the RNA
  4. Decant upper aqueous phase into fresh eppy
  5. Add 0.5mL isopropanol (RNA may visibly precipitate at this point),10 mins RT
  6. Spin 12000g at 4 degrees for 10 mins (should see RNA pellet), discard supe
  7. Wash with 1mL 70% ethanol (vortex and spin at 7000g at 4 degrees for 5 mins)
  8. Airdry RNA pellet until almost dry (completely dry pellet is harder to resuspend)
  9. Now resuspend in DEPC water, or continue with... Isolation of poly A+ RNA
  10. Resuspend in 1mL STE with proteinase K and SDS:
STE (0.1 M NaCl, 10 mM Tris 7.4, 1 mM EDTA) 50 ml
- 1mL 5M NaCL stock
- 0.5mL 1M 10mM Tris 7.4 stock
- 0.1mL 0.5M EDTA stock
- 48.4mL dwater
STE with 200痢/mL proteinase K and 0.5% SDS:

To 10mL STE add:

- 100無 of 20mg/ml proteinase K stock
- 250無 of 20% SDS stock
  1. 10 mins RT (try to resuspend the pellet at this time) 1 hour at 60 degrees
  2. 10 mins RT (all RNA into solution by now)
  3. Make up to 0.5M NaCl (add 110無 5M stock), spin out any debris
  4. Add ca. 200無 (packed volume) oligo(dT)-cellulose slurry in STE (0.5M NaCl)
  5. Wheel 10 mins RT
  6. Pulse spin and discard supe, resuspend in 1mL binding buffer
  7. Repeat binding buffer wash twice, transfer suspension to econocolumn
    1mL washes with binding buffer
    2mL wash with washing buffer
  8. Elute with 2 x 200無 elution buffer
  9. Set aside 50無 for quantitation, adding to this 50無 of elution buffer
    Read OD 260nm against elution buffer blank
    To remaining 350無, add 35 無 5M NaCl and 800無 ethanol, store in freezer.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998