Transfections questions - (Apr/09/2008 )

Hey everyone,
I have a question about trasnfections into Ecoli. After transformation most protocols tell you to shake cells at 37 for 1 hr with 1 ml LB or CG than plate.
I have not been performing this set but instead I just plate directly after trasnformation (on ice for 1hr). My transformations seem to be going fine (colonies grow). So is the 1 hr incubation step really necessary? I'm selection with amp. If I select with another antibiotic will is still be ok to skip the incubation step.

Thanks

The incubation step is to get the cells growing and regenerate the membrane before plating, it can be skipped if you have to, but it is best to do it, especially if you are using some of the more cytotoxic selection agents, or ones that require dissolving in substances other than water.

The 1 hr incubation is for antibiotic resistance gene to be produced by E.Coli.

I think it might be difficult for kanamycin plasmids to grow if they are not incubated.

if you are transforming with circular plasmid, you can skip this step and do fast transformation. I wouldn' t do it if I'm doing transformation with ligation reaction

Ampicillin is bacteriostatic rather than bacteriocidal. Plating out amp transformants will work without outgrowth. This is not true with most other antibiotics. I'd suggest 1 hour. We've found that with tet selection, outgrowth of 1.5 to 2 hours definitely improves transformation efficiency by quite a bit. This much less important with Kan and chloramphenicol.