Gel Purification -- Anyone else have this problem?

Warren Lushia walush0 at ukcc.uky.edu
Sun May 18 21:45:33 EST 1997


Hello molbio experts,
Our lab has had a reoccuring problem (nightmare) with gel purification 
and subsequent ligation of fragments using a couple of commercially 
available kits (Geneclean and Qiaex gel extraction kit).  We first 
noticed this with the Qiaex kit several months ago.  The kit worked 
extremely well at first, but about half way through the reagents, we 
experienced a problem in which we could not get any of the clones we 
wanted.....this was using all different vectors and inserts (PCR 
generated or restriction fragments) from "difficult" clones (IE 2 blunt 
ends) to very easy clones (2 different sticky ends)....the results were 
very consistent...all junk.  We would receive clones, but they were not 
correct, and it appeared as though there had possibly been a deletion of 
part or parts of the insert (both from sequencing and restriction 
analysis).  After much troubleshooting in which we tried everything from 
different suppliers of ligase to praying to the cloning gods, we finally 
narrowed the problem to one of the buffers (QX1) supplied with the kit.  
Luckily the buffers were supplied in 2 separate aliquots or we may have 
never figured out what was wrong.....and the difference was like night 
and day, all of a sudden everything worked for everybody.  Since many 
people use this kit, it was hard to say what might have happened, so we 
just attributed it to someone screwing up the buffer.  I should also 
mention yield was fine (as measured by running an aliquot on a gel) 
whether or not we had the cloning difficulties, and the composition of 
the buffer is a "trade secret" so I can only say it is some sort of 
chaotropic salt.  

We then decided to give the Geneclean system a shot.  The theory of the 
kit is almost identical.  However, recently, about 3/4 the way through 
the resin provided with the kit, we are having identical problems as we 
had with the Qiagen kit, ie, no correct clones over many (>20) attempts 
using all different vectors and inserts.  The buffers in this kit are 
single aliquots so I can not troubleshoot if there is something wrong 
with the kit.

My question is: has anybody else experienced similar problems?  Is there 
some buffer or reagent that can go bad? (All the kits were relatively 
new ie, <6 months)  Is there some special precautions other than storing 
at the recommended conditions and using fresh tips when using these 
buffers?  The solution is obvious -- find a different protocol or just 
buy a new kit, but I am very curious to know what is going wrong and 
if/how it can be prevented in the future.  I am fairly certain it is not 
sloppy lab practice by anyone, as it screws up everybody in the lab (ok, 
so that is only circumstantial evidence, so you'll have to trust me :)

Any thoughts or suggestions are most welcome.

Thanks, Warren..



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