DNA recovery by phenol/chloroform extraction or GeneClean

William Coetzee coetzeew at is2.nyu.edu
Tue Feb 18 19:46:08 EST 1997


We observed something strange recently. We restricted a vector with
NcoI/BamHI and then gel-purified a fragment of about 3kb (GTG
agarose/TBA followed by Qiagen Gel column purification. A subsequent
mini-gel showed a strong 6kb band and a faint 3 kb band. Why? Is it
possible that spontaneous ligation occurred?

William

Chris Boyd wrote:
> 
> Wilson (netson at uxmail.ust.hk) wrote:
> 
> (snip)
> 
> : I have heard that if DNA with stickly end(s) (digested by restriction
> : enzyme e.g. EcoRI) was vortexed, they will stick together and
> : consequently inhibit ligation.  This is screw up of DNA,  is it possible
> : to occurred?
> 
> I don't believe so.  For a start, how could you prove this idea? It's
> not sufficient to show that vortexing inhibits ligation, which it could
> do if the DNA gets sheared in the process (though shearing problems
> only kick in significantly with large (10s of kbs) linear molecules).
> 
> Best wishes,
> --
> Chris Boyd                       | from, | MRC Human Genetics Unit
> chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
> http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND



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