Subcloning: Loosing my favorite gene

Allan via methods%40net.bio.net (by allan.debono from gmail.com)
Sat Feb 10 18:50:02 EST 2007


Hello everyone,

I'm hoping for some advice.  I have cloned a gene into pUC19 and have
sequenced.  It is perfect.  I have linearized it at an obscure cut
site (SgrAI) and phosphatase treated the resultant 5' protrusions with
Antarctic phosphatase.  The linearized product runs at the desired
size (6.3 KB).  Upon ligating GFP with sticky ends complimentary to
the cut site in the gene  (GFP is cut out from a cloning vector
construct) I get transformants.  I get no transformants on my negative
control:  linearized, phosphatased gene of interest.

Now, upon restriction digestion with KpnI I should get  two products:
pUC19 (2.7 KB) and my favorite gene+cYFP (4.5 KB) I ONLY GET a single
3 KB band.  After some fighting with this problem I sequenced the
resultant 3 KB plasmids.  They did not contain my gene.  They
contained pUC19.

So it appears that I am getting a massive deletion.  Does this sort of
thing happen frequently?  Given how easy it was to clone the gene I am
in disbelief how difficult it has been to introduce the ~800bp GFP.

Thank you,

A



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