pEGFP-C1 transformation

Carnac carnac at realitys-edge.com
Sat Dec 21 16:47:44 EST 2002


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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