EST sequencing strategy

lmc at sevax2 lmc at sevax2
Thu Aug 31 02:49:38 EST 1995

In article <DE27w7.6JK at news.nsw.CSIRO.AU>, D.Adelson at (David Adelson) writes:
> We are about to start looking for some sheep tissue 
> specific ESTs, using a lambda ZAP cDNA library.
> > Is it possible/better to use PCR to amplify the insert
> regions of individual clones and then cycle sequence
> the PCR product directly?  We are looking to avoid having
> to do the in vivo excision, plating and miniprepping to
> get template for the sequencer.

The method described in this paper is very reliable
"Large-scale production of DNA sequencing templates by 
microtitre format PCR" Rosenthal, A., Coutelle, O., Craxton, M.
Nucleic Acids Res. 21, 173-174, 1993.

It describes methods for bacteria and M13 but I guess it could
also be applied to your specific problem.


Luis Corrochano
Departamento de Genetica
Universidad de Sevilla
Apdo. 1095
E-41080 Sevilla
e-mail LMC at

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