extracting blue dextran and leaving behind DNA?

Richard Converse Richard.Converse at UC.EDU
Fri Mar 14 16:00:23 EST 1997


Hi everyone!

	I had a question for you: I have an end-labelled S1 probe which I want
to purify from unincorporated nucleotides and small lambda exonuclease
digestion products. I am using a 2mL G25 column and putting BPB and blue
dextran in the sample before loading. Since the probe comes out with the
blue dextran, the correct fractions are easy to collect. The problem is
that since I want to re-label the probe after my 1st column run, I would
like to know if blue dextran can (or even needs to) be removed from the
collected fractions before re-labelling. 

	The reason I am re-labelling is that my first PNK labelling is done in
lambda exonuclease buffer for the purposes of being able to follow the
probe on the column--I don't want to precipitate the ssDNA probe because
I am scared of losing it (~1000bp)--so I figured I would just pre-label
it, run the column, collect the fractions, re-label it, and run it
through the column again. I would dialyse or amicon concentrate the
probe except that I suspect that the leftover DNA fragments from the
lambda exonuclease digestion will reduce the efficiency of the PNK
label--so I figured that the G25 column would kill 2 birds with one
stone--remove the smaller DNA oligos and the unincorporated nucleotide
from the full-length S1 probe in the 1st run, and remove the
unincorporated nucleotides alone in the 2nd run.


							e-mail or newspost me  
							if you have any input!


							Richard--



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