To all the people that might help resolve this strange problem!
I have some trouble with the BamHI-BglII fragment of pNKY51! This is the
plasmid containing the hiG sequences of Salmonella flanking the URA3 gene
and was contructed by Alani et al.(Genetics 116:541) In order to make a
knock out of the gene I'm working on, I would like to put it in a more
convainient vector (that is pBluscript) using the BamHI site.
OK! It seems easy! But I'm unable to obtain it! I start with the 3,8kb
fragment of pNKY51 (gel purified) and pSK(BamHI, CIAP and gel purified) and
all transformant I screen do contain a plasmid, but which do not contain
the pSK sequences! In fact, when I cut it with two enzymes who should cut on
both side of the insert, what is generated is a fragment of MW around 4-4.2
Is there somebody somewhere who can help me to solve this very strange
problem? The hisG-URA3-hisG by itself is stable in E.coli since I have
been able to subclone it already in the EcoRV site of the same pSK. Is
there something that I don't know about the pNKY51?
Waiting for your hints,