subcloning the hisG-URA3-hisG of pNKY51?
Tue Feb 28 11:40:38 EST 1995
In article <950225.215439.EST.3000JGER at vm1.ulaval.ca>,
3000JGER at VM1.ULAVAL.CA (Julie Gervais) wrote:
> 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?
I suggest that you might be getting interference from an unwanted
fragment og pNKY51. Digestion of pNKY51 with Bgl-Bam produces
TWO 3.8 kb bands. the one you dont want contains origin seq., and should
be cut down with pvuII prior to gel isolation.
sound like your problem?
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