Cloning low abundance PCR products?
leach at mbcrr.harvard.edu
Thu Apr 14 00:33:19 EST 1994
In article <1994Apr12.114758.8694 at mbcf>, neale at mbcf.stjude.org wrote:
> Does anyone have some good ideas regarding cloning **small** amounts of PCR
> We currently have an assay that generates 3-6 fragments that we wish to
> sequence. These fragments are in low abundance, and can only be detected by
> hybridization with an internal oligonucleotide probe (usually over weekend
> exposure required).
> On agarose gels there is a smear of potential PCR products by ethidium
> staining. (We are amplifying from genomic DNA). When we take the whole reaction
> and clone via T-vectors (Promega gives good results in our hands), we find no
> clones that hybridize with our internal probe. Usually we screen 500-1000 white
> colonies per time.
> We have tried to gel purify the bands after freezing the gel while doing the
> autoradiography, but have been unable to enrich the products that way.
> Any suggestions or hints would be welcomed.
Perform re-pcr or even better nested-pcr on purified fragments or on a ul
of the first pcr reaction. Then shot-gun clone purified fragments or pcr
reaction into t-vector.
I have done this for inverse pcr when multiple bands were observed and i
didnt know which one i wanted.
..... Martin Leach Email:leach at mbcrr.harvard.edu
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