cloning problems

hl15 at hubrecht.nl hl15 at hubrecht.nl
Tue Sep 27 12:05:31 EST 1994


Hi,
I am trying to subclone a number of DNA fragments from a cosmid
into bluescript. I have dephosphorylated the ends of the vector. A
ligase control (ligase*vector) gives zero colonies. To be sure I have
done a ligase*fragment control, again, no colonies. Now I combine
the fragment and the vector and ligase and get many colonies.
Looking good? No, all colonies contain just the bluescript plasmid.
I have done these experiments plus controls several times over with
the same result. I have the problem with several fragments (ClaI-
ClaI) but only if they are longer than 6 kb. Perhaps I have the basic
problem always but is it only visible when efficiency drops with
larger fragments. It is as if the inserts are specifically removed from
the plasmid. It is driving me pretty much crazy

(on behalf of)
Isabelle





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