No colonies with site-directed mutagenic plasmids - (May/25/2005 )

Hi all,

I am having problems with E. coli transformation. I am trying to transform them with different site-directed mutagenic plasmids, as I have done successfully until now, but I don't get colonies growth after overnigth incubation at 37 ºC.
The procedure is correct since control plasmid goes well. The problem is in the mutated plasmid, but why? Plasmid amount is OK as seen in gel. I add 4 microlitres of mutated plasmid to bacteria.

Any ideas will be very appreciated

Thanks

Hi,

Have you tried running a small amount (~10µl) of your site-directed PCR product on a gel? Check if you can see band... maybe your PCR isn't working.

I am using a the Stratagene kit. Never had any problems.

Good luck.
Freiberger