Help wanted. Direct sequencing Lambda

gaitken at chmeds.ac.nz gaitken at chmeds.ac.nz
Sun Jun 19 18:27:42 EST 1994


I have been trying to direct cycle sequence a lambda clone from a clontech
genomic library. 
The clone is positive, for the gene of interest, by colony hybridisation and 
PCR with gene specific primers.
Several attempts with various primers using stratagenes 'cyclist'and 'exo-
pfu' kits on purified lambda DNA have failed.
The template used was 30 fmol of purified large scale lysate DNA. The clone
has a 15kb insert positive for the gene of interest.
I have determined the insert size recently by the 'Barnes long extension
PCR method.
Noting stratagenes claim of being able to direct sequence an eluted single
plaque with their kit has anyone got any helpful hints or comments?
Has anyone successfully direct sequenced a single plaque by this method?

Any assistance will be greatly appreciated
Geoff Aitken    



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