PCR purification kit

Zhonglin Chai zhonglin.chai at med.monash.edu.au
Mon May 22 02:13:46 EST 2000



ChenHA wrote:

> I wonder if anyone out who can recommend a good purification
> kit for pcr product.  I have been using the Qiaquick pcr
> purification kit, but have not been satisfied with the
> result.  In my experience the yield is poor with the kit,
> sometimes only 10% of the pcr product is retrieved.  In the
> past I have ignored this deficiency with the kit because it
> is fast and convenient, but surely there is a better one out
> there?

    Depending on what you will use the purified pcr products for, you
may have a varieties of choices there.
    Personally I have been using Promega's Sepharcryl S400 matrix to
purify or to get rid of primers of pcr products for sequencing. You just
need to load the matrix to a spin column, and spin to dry the matrix,
and then load pcr reactions (30uL or a bit more) to the surface of the
matrix, and spin again for a few minutes to collect the DNA. Anything
smaller than 400bp will retain in the column and the larger products
flow through. You then just need 2-5uL directly for sequencing. They
have also S300, namely able to get rid of DNA smaller than 300 bp. This
was initially designed to get rid of small synthesized cDNA fragments
for library construction, but it worked very well for pcr products
purification for sequencing purpose in my hands.
    If you need to clone the pcr products, you may need to get rid of
the DNA polymerase and other junks as well. Either phenol/chloroform
extraction or gel purification before restriction digestion seems to be
necessary and worked equally well in my hands. Of course, after
digestion you need to gel purify before doing ligation. I have been
using Qiagen's Qiaquick Gel Extraction kit that works beautifully with
satisfactory yield. BTW, elute the DNA in 10mM Tris solution provided in
the kit. If you elute in water, either the yield may drop, or the DNA
may migrate differently in agarose gel showing one or more additional
bands with smaller molecular weights than expected, although the MW may
become back to normal after adding tris or salt to the solution.

--
ZhongLin Chai, PhD
__________________________________________________________
Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital
Commercial Rd, Prahran, VIC 3181, AUSTRALIA
Telephone: (61 3) 9903 0698 (lab)
           (61 3) 9903 0696 (office)
Mobile:    0413 58 1940 or International: +61 413 58 1940
Fax:       (61 3) 9903 0731
email:     zhonglin.chai at med.monash.edu.au
___________________________________________________________






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