Gel Purification -- Anyone else have this problem?

brett brett at BORCIM.WUSTL.EDU
Mon May 19 08:47:04 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..


I doubt someone is contaminating your kit with say, a nuclease, or other
enzyme, as I imagine these kits use guanidinium salts to clean up your
samples. Is it
possible that your reagents are contaminated with a plasmid? It might be good
to compare notes among lab members as to what products were obtained. Also,
based on Raj Mehta's and Sailesh Surapureddi's notes, maybe find out if they
characterized their bozo clones. You may have basis for a complaint against
the kit manufacturer. Seems like a lot of wasted time for a product which is
supposed to simplify your life. I mean, you could have made up your own kit.
Then you'd only have yourself to blame for your problems. My 2c.
Brett Lindenbach
Dept Molecular Microbiology
Washington University School of Medicine
St Louis, MO

"I own my own pet virus,
I get to pet and name her." - K Cobain




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