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subcloning the hisG-URA3-hisG of pNKY51?

Julie Gervais 3000JGER at VM1.ULAVAL.CA
Sat Feb 25 21:53:24 EST 1995


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
  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?

  Waiting for your hints,
  S.Tremblay
  Universite LAVAL
  Quebec



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