multiplying bands in gel purification

T.S. Grewal tsg1 at york.ac.uk
Thu Feb 20 08:31:51 EST 1997


In article <330B5256.5AD1 at is2.nyu.edu>, coetzeew at is2.nyu.edu wrote:

I also had the same problem. Quiagen said it was due to denaturing of the
ds PCR product into ssDNA ??!!
So they recommended heating the purified PCR to 95 C and allowing it to
cool slowly at room temp.
This did not work for my "purified" PCR -but you could give it ago. I
found that if I used the GeneClean
II or III kit I got 1 single band of the correcy size. After that the
Quiagen PCR purification kit has remained
unused !!! Also, the yields from the Quiagen kit were really poor!!!


> John G. Luz wrote:
> > 
> > Folks--I'm tearing my hair out.  I cut a nice clean tight band out of a
> > gel, and purified it with the qiagen gel extraction kit.  But when I ran
> > the gel-purified band out on a gel to check my yield, I got two bands,
> > both about equimolar.  One band looked about the right size.  The other
> > looked about 1kb smaller.  The band I gel-purified was 5.4 kb and cut
> > with NdeI and Xho.  What's going on?--John
> 
> We also had some strange results with the Qiagen gel extraction kit. See
> my post "Subject: DNA recovery by phenol/chloroform extraction or
> GeneClean". Repeated here for convenience:
> 
>     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 Coetzee

-- 
T. S. Grewal
Bone & Joint Research Group
Department of Biology,
University of York,
York,  UK.

email: tsg1 at york.ac.uk



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