Gel Purification -- Anyone else have this problem?

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Wed May 21 10:35:37 EST 1997


This is exactly why I stopped using these gel extraction kits.  I
abandoned them after I found this spin-out technique.  It has worked
well and reliably for me in the past year that I have used it.
The reference is (biotechniques 17(4) p634 1994)  Chuang and Blattner
Ultrafast recovery of DNA by centrifugation thru a paper slurry.  
Basically, you make a paper slurry by cutting up a 10x10 piece of 3mm
paper (like the one used to back gels for drying), place the pieces in
a
50 ml tube with 40ml of TE and shake like hell for 5 min or untill the
paper disperses.  Store at 4C.  Punch a hole in the top and bottom of
a
0.65ml tube with a 25ga meedle or equivalent pointy object (I use a
forcep).  Fill the tube with slurry, cap, place in 2ml tube and quick
spin
to elute TE and settle paper.  Tamp down the paper to the bottom with
a
glass rod or flamed pipette tip.  Spin again for 5 min to elute
remaining
TE.  Place 0.65ml tube inside a fresh 1.5ml tube and load the gel
slice
into the smaller tube.  Spin for 10-15min at full speed.  Discard the
0.65ml tube and save the eluate.  I have ligated this directly and it
has
worked on most occasions.  Minimize the excess agarose around your
band to
maximize the concentration of DNA in your eluate.  Yield is around
50-80%, its fast, easy and cheap (and the best gel extraction method I
have come across so far).  By the way, freezing the gel slice before
centrifugation dramatically REDUCES yield in my hands (i.e.
"freeze-squeeze" sucks, and "just-squeeze" works great). Good luck.

                                                Regards         SVEN




In article <199705191345.IAA07747 at borcim.wustl.edu>, brett at BORCIM.WUSTL.EDU (brett) writes:
>>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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