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RESEARCH DIVISION Laboratory Manual

 


 

Poly-A+ RNA Preparation Using Proteinase K and SDS

NOTE: All solutions should be autoclaved or treated with 0.1% DEPC. Homogenizer probe should be treated with 0.5M NaOH for about 5 min, then rinsed with sterile H20 and STE. Econo column should be washed with 0.5M NaOH then several aliquot’s sterile H20.

  1. Wash cells with PBS.
  2. Resuspend thoroughly at 107/ml in STE (0.1M, NaCl, 10mM Tris 7.4, 1mM EDTA), predigested proteinase K to 200g/ml and SDS to 0.5% and homogenise with an ultraturax. For mouse tissues (one brain or 10-15 E13 embryos) homogenise in 10ml STE with 1mg/ml proteinase K, 0.5%SDS in a 50ml tube and then top up to 40ml with STE, 0.5% SDS (PK final 200ug/ml).
  3. Incubate 60C 1h then 37C 20'. Make up to 0.5M with respect to NaCl and for tissues spin 3.5K 20C for 10min. Transfer with a pipette to a fresh tube.
  4. Add oligo dT-cellulose suspension (typically 0.25ml packed volume) and place on wheel for 1 hour. [To prepare oligo-dT ad to a 10ml tube, wash in 0.1M NaOH and spin down 2' in bench top 3K. Wash twice in binding buffer (0.5M NaCl, 10mM Tris 7.4, 1mM EDTA, 0.1% SDS) and make to a 50% slurry and add to lysate. Resuspend for each sample as the oligodT settles fast in a pipette.]
  5. Spin down oligo-dT in lysate and wash twice with 15ml binding buffer. Transfer to alkali-treated (ie. RNASE-free) econo-column.
  6. Wash with at least 10ml of binding buffer. Wash with 2ml wash buffer (0.1M NaCl, 10mM Tris 7.4, 1mM EDTA, 0.1% SDS).
  7. Elute RNA with 2 x 0.4ml of elution buffer (10mM Tris pH 7.4, 0.1 mM EDTA) into an ependorf tube. Vortex and remove 50ul for OD. Split sample into two ependorf tubes and add 1.0ml ethanol and35ul 5M NaCl. Freeze at -20C.

This method can also be used for solid tissues. There is no hard rule for how much material to use, but since even a small piece of tissue is equivalent to a large number of cells, it is advisable to use a large volume of STE/proteinase K/SDS. The procedure works well for adult brain, mouse embryos and cell lines but not very well for other adult tissues. Acid phenol is the best technique for the latter.

Solutions

  100 ml 400 ml
H2O Tris 2M NaCl 5M EDTA 0.25M SDS 10% H2O Tris 2M NaCl 5M EDTA 0.25M SDS 10%
Bind 88 0.5 10 0.4 1 352 2 40 1.6 4
Wash 96 0.5 2.0 0.4 1 384 2 8 1.6 4
Elute 98 0.5 -- 0.4 -- 392 2 -- 1.6 --
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