phenol:chloroform extractioon and chloroform extraction

Lesley Collins L.J.Collins at massey.ac.nz
Fri Mar 8 21:07:02 EST 1996


Hi!

My name is Lesley Collins and I work with DNA preparations of bacterial 
plasmid DNA.

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.
Lesley




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