shotgun cloning: gel shows inserts but only plasmid sequence obtained - (Oct/15/2007 )
I am doing shotgun cloning of a poxvirus. I have digested the viral DNA with HindIII and cloned it into pUC21. After digestion with HindIII and running on a gel, there are inserts present ranging in size from 500bp to 3kb. More than 50% of the samples that I sent off for sequencing show homology only to pUC when I BLAST the sequence. Any ideas on how this could happen? Thanks.
-yellowish-
The primer you are using is directed away from the cloning site? You picked colonies that have more than one kind of plasmid, and the prep has mostly the non-insert plasmid? This would show an insert from the rare plasmids on a gel, but sequence as a no-insert plasmid.
-phage434-