Subcloning: Loosing my favorite gene

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"peter" <peter.ianakiev from gmail.com> wrote in message 
news:1171204160.599253.21680 from v33g2000cwv.googlegroups.com...
> 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.

Ligate gene + reporter gene first, and then ligate the product into your 
vector backbone.

>> 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.

Large deletions do not happen frequently. You just need to check each step 
before you do the next step.

ST