dk at no.email.thankstospam.net
Sat Dec 21 18:21:39 EST 2002
In article <au2nlk$71g$1 at bunyip.cc.uq.edu.au>, "Carnac" <carnac at realitys-edge.com> wrote:
>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?
Are competent cells really competent? What's the transformation efficiency
with control plasmid? Dis you do a control to make sure ligase works too?
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