pEGFP-C1 transformation

EK khatipovNO-SPAM at NO-SPAMuchicago.edu
Mon Dec 23 13:53:08 EST 2002


pEGFP-C1 (Clontech, encoding EGFP with C-terminal His6 tag) encodes for
beta-lactamase (ampicillin resistance). I wonder why even a few colonies
survived on the kanamycin plates. Probably your E.coli stocks are not pure,
or you flame-dried your plates for too long :-)
Emir

"Carnac" <carnac at realitys-edge.com> wrote in message
news:au2nlk$71g$1 at bunyip.cc.uq.edu.au...
> Hey, all.  :)
>
> I am currently attempting to transform electrocompetent cells with a
> pEGFP-C1 vector/insert plasmid, and am having very limited success - in
the
> range of absolutely no colonies, to very few.  I'm using a 1 uL volume of
> plasmid to 20 uL of competent cells in a 1 mm cuvette.  The plasmid  and
> insert digest was with SAL-I and Bam-HI, where the SAL digest was
performed
> for 2 hours prior to the adding of the BAM enzyme, for a further hour.
The
> cut plasmid plus insert were then ligated with T4 ligase at room
temperature
> for 1 hour prior to transformation.  I used a 3:1 insert:vector ratio for
> recombination.  After transformation, the cells were iced for 5 minutes,
and
> then 1 mL of LB was adding.  The cells were transferred out of the
cuvette,
> and allowed to recover for 1 hour, prior to plating on media containing
> kanamycin.  The plate was then dried under a flame for 20 minutes.
>
> Has anybody else had any problems transforming E coli with this plasmid?
>
>
>





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