subcloning the hisG-URA3-hisG of pNKY51?

Brian Ernsting at *
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
>   kilobases.
> 
>    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?
 

good luck, 
  
brian



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