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 :-)
"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
> 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
> for 2 hours prior to the adding of the BAM enzyme, for a further hour.
> cut plasmid plus insert were then ligated with T4 ligase at room
> 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,
> then 1 mL of LB was adding. The cells were transferred out of the
> 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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