Polyethyleneimine precipitation protocol

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Fri Nov 19 19:09:27 EST 1999


On Sun, 14 Nov 1999 20:30:57, Kulikov Eugene <eumenius at imb.ac.ru> wrote:

ð Hello all,
ð 
ð I recently began to produce recombinant Taq-polymerase and the first
ð problem I have reached is purification of enzyme of DNA traces. Does
ð somebody know the protocol of DNA precipitation with polyethyleneimine
ð (Polymine P)?

Make a solution of 10% Polymin P in pH 8.0 Tris buffer (or Hepes pH 7.0, 
whichever is more appropriate & compatible with your isolation buffer).

Dialyze 4 hr to o/n vs. same buffer.

Method 1) Bring protein extract to a salt concentration at which the 
polymerase stays in the supt after PEI pptn. You could use ammonium sulfate 
for this. 

Add PEI, dropwise with stirring (on ice in cold, if appropriate), to your 
protein solution. Final concentrations used are usually 0.1% to 1.0%. Continue
to stir for 30 min. Centrifuge 20 to 30 min at 10,000 rpm. Supt will heve 
soluble enzyme; pellet will have DNA, large RNA, ribosomes, membranes 
(phospholipids). 

Concentrate protein from supt with A.S. ppt'n, or by binding to and elution 
from P-cell or other cation-exchanger, or via His-tag, etc. 

Method 2) Exactly like method 1; use this IF the Taq polymerase binds tightly 
to endogenous high-mol-wt DNA. (You can try this on a small scale first.) Do 
the PEI pptn at low salt conc so the pol stays bound to the DNA. Spin as 
above. Elute the pol from the pellet by extraction with ammonium sulfate 
solution. 

References: old paper by Dick Burgess on purification of RNA polymerase? and 
of (I think) DNA topoisomerase from wheat germ; Gegenheimer, Meth Enz 182 
[42].

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