Insert seems to stimulate self ligation of pGEX vector using EcoRI single digest method
dk at no.email.thankstospam.net
Sat Aug 24 11:44:12 EST 2002
hansw at sci.kun.nl (Hans Wessels) wrote:
>I have encountered a problem i cannot quite comprehend. I have tried
>to ligate a gene into the pGEX 4T1 vector by means of single digest
>with EcoRI. I will try to explain the method and the resulting problem
>so that you can try to help me out.
>After screening of 60 CFU's by doing a PCR on the CFU's
>using vector primers it showed that NONE of the 60 CFU screened
>contained the gene but DID CONTAIN the vector. Recall that everywhere
>along this experiment everything was done using mastermixes; so
>differences in the ligation and transformation solutions/cells cannot
>acount for the result.
The most likely explanation IMHO:
Because vector was dephosphorylated and a number of colonies was
small , there is really no way something in your mix could make
the vector to self ligate (unless you added kinase somehow).
Therefore, your ligation worked fine but well after transformation -
(when amp reistance was already acquired from a circular plasmid)
the bugs threw away your gene.
Why exactly this happened I don't know. Your insert might be toxic.
Or you might have had almost exclusively concatamers ligated in
and they were excised by cells.
I'd try directional cloning and see if it works. Also, sometimes changing
strain helps with insert stability problems.
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