Gel Purification -- Anyone else have this problem?

Warren Lushia walush0 at ukcc.uky.edu
Mon May 19 18:33:08 EST 1997


Thanks for all the response so far.  In my original post, in the 
interest of brevity, I did not include all the potential problems we had 
eliminated as possibilities for the strange results (before we discover 
it was one of the buffers).  We did try several strains of bacteria 
(TG-1, JM101, DH-5a) but in our case we were relatively certain this was 
not going to make a difference, and it didn't.  The buffers are not 
contaminated with any plasmid.  In one case, sequencing was done by 
another person in my lab, and he found that along with some correct 
clones, most of the clones contained deletions of anywhere from a few 
bases to several hundred.  However, in that case the product was 
generated by PCR, so we can not be sure the problem was not in the PCR.  
But this lead to my current feeling of the ends of the DNA being somehow 
"modified" during the gel extraction procedure, although I do not know 
if this is a reasonable possibility.  After we began using the new 
aliquot of buffer (as mentioned in my previous post) the mysterious 
problems of the deletions I mentioned disappeared.  Also, one additional 
point -- although, as I mentioned, yield was fine, the number of 
transformed colonies tends to be substantially lower than you would 
expect for any given clone.  It may sound like DNase contamination, but 
I would be very amazed if someone contaminated one of the buffers with 
DNase (especially enough to cause so many problems).  Anyway, thanks for 
all the responses, Warren..



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