Subcloning: Loosing my favorite gene
(by customer from newsnet.com)
Tue Feb 13 07:36:44 EST 2007
"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
>> 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.
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