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.
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.