Cloning low abundance PCR products?

Martin Leach 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
> 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.


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
-- 

.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323        
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329         
 _/  |-/__==/  80 E. Concord St. (L603)
(BULLDOZER) \_ Boston MA 02118            "Not the old underpants on your
               USA                           head.....WIBBLE" -BLACKADDER 
p.s. try BioMOO (virtual biology on the internet - telnet
bioinfo.weizmann.ac.il 8888)
 



More information about the Methods mailing list