Cloning low abundance PCR products?

neale at mbcf.stjude.org neale at mbcf.stjude.org
Tue Apr 12 12:47:58 EST 1994


Does anyone have some good ideas regarding cloning **small** amounts of PCR
products?

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.

Geoff Neale

Dept. of Virology and Molecular Biology        Internet: neale at mbcf.stjude.org
St. Jude Children's Research Hospital             Phone: (901) 522-0400
Memphis, TN                                         Fax: (901) 523-2622





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