PCR purification KITS: Best one??????

Mr. Chris Hoyle bmbckh at biovax.leeds.ac.uk
Thu Aug 14 07:24:03 EST 1997


In article <33F20C59.C56 at bioc.rice.edu>
Mike Lyristis <mikel at bioc.rice.edu> wrote:

> Can anyone help me... I am at that point. I perform PCR reactions and I
> get one specific product. I have used the Qiagen clean up kit, the
> Geneclean Kit, and I have done a good ol ammonium acetate precipitation.
> What I see is conversion to what presumably is a single stranded form. I
> have repeated the purification and I get the same result consistently. I
> rang the Qiagen reps and they told me that for DNA that has some unique
> characteristic (such as high A+T content), there is some conversion to a
> single stranded form. I am having an immense amount of trouble with it.
> I am also wasting a lot of time with trouble shooting this problem which
> exists for 4 independent PCR reactions.
> 
> Has anyone any suggestions that may be of some help. I do not know what
> to do...
> 

I routinely isolate the desired PCR product by agarose gel
electrophoresis and excision of the appropriate band. AGE is required
because other minor, non-desirable products are produced by PCR. The
product is then extracted from the agarose using a gel extraction kit
from Qiagen. The couple of occasions that PCR has produced a single
product I cleaned the DNA up by phenol/chloroform extraction and
precipitation in ice-cold isopropyl alcohol. DNA was pelleted,  dried
and resuspended in sterile milliQ-water. The PCR product was suitably
clean for sub-cloning into an appropriate vector.

I hope this helps.
------------------------------------------------------
Chris Hoyle
Department of Biochemistry and Molecular Biology
University of Leeds             phone +44 0113 2333172
Leeds LS2 9JT                   FAX   +44 0113 2333167
Great Britain           e-mail BMBCKH at biovax.leeds.ac.uk
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