phenol:chloroform extractioon and chloroform extraction
L.J.Collins at massey.ac.nz
Fri Mar 8 21:07:02 EST 1996
My name is Lesley Collins and I work with DNA preparations of bacterial
The proceedure that I use goes as follows:
Ensure that your DNA solution is in at least 100 microlitres.
The procedure here is for volumes of 100 - 500 microlitres.
Add an equal volume of phenol/chloroform:iso amy alcohol
Vortex or invert briefly to mix
Centrifuge at 12 - 13 000 rpm in a microfuge.
Transfer the aqueous (non-phenol) layer to a fresh microfuge tube.
Add an equal volume of chloroform:iso amyl alcohol (24:1)
Centrifige as above.
Transfer the aqueous phase to a fresh microfuge tube.
Usually this is the top phase.
After this precipitate the DNA using ether Sodium acetate or ammmonium
acetate and ethanol.
The phenol we use has been equilibrated to pH8.0 with 50 mM TrisHCl
The chloroform is mixed in a ratio of 24:1 with iso amyl alcohol.
if you need to know the procedures for preparing the phenol etc.
then please let me know.
L.J.Collins at massey.ac.nz or Lesley.Collins at nzdri.org.nz
All the best.
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