Subcloning: Loosing my favorite gene

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Sun Feb 11 09:29:20 EST 2007


On Feb 10, 6:50 pm, "Allan" <allan.deb... from gmail.com> wrote:
> 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

Hi Allan,
Happens sometimes, especially if you force the ligation. Try growing
bacteria at 30oC, that might help. Also, to have full idea what is
going on, I will suggest that you make the following contrtols: Vector
+ Ligase, Vector + Ligase + Kinase, Vector + insert + No Ligase. Hope
this will help,
Peter



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