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Help: Subcloning

Sergio sergioal at solea.quim.ucm.es
Tue Jan 18 06:54:52 EST 2000



David Antonetti wrote:

> We are subcloning two separate PCR products for different cDNAs.  We
> have ligated and transformed both inserts into 3 different vectors and
> two
> differenct e coli lines and get the same result.  We obtain amp
> resistant colonies but we cannot retrieve any plasmid DNA using either
> alkaline lysis
> and phenol chloroform or using qiagen midi  kit.  However, if we try to
> PCR from the plasmid prep we obtain product with the correct insert.
> Other plasmids prepared in parrallel are fine.  Anyone with any
> suggestions?
>
> David Antonetti
> dantonetti at psu.edu

Just a very simple checking:
Have you check the competent cells you are using?? Try to plate them before the
transformation, maybe there is an AmpR contamination.
Ah, check the plates as well, i had similar problems and finally it was an old
ampicillin.

Sergio





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