3 replies to this topic

[rameshcdri]

Hi Guys, I am new member of this forum. I am working on Pseudomonas aeruginosa.

I did electroporation of the non-replicative plasmid pEX18apGD by using 10 min protocol. After 2 and half hour recovery incubation, I plated 500ul to Gen selective plate (30ug/ml; without centrifuge). After 24 hr, there was no colony,  but after 48 hr there some very small colonies appeared. Are these real colonies? or non specific colonies due to breaking down of Gent. antibiotics. The non-selective plate also have same texture of colonies.
Do you suggest me to go further to screen the colony of interest?

[pito]

well, I would check them to be sure, but the changes are high they do not contain an insert.

But on the other hand: the non selective plates have the same kind of colonies? Then it might just be ok.... but are you sure you are growing them like you should, because you should see nice colonies normally.

[Biouday]

My suggestion is don't waste time in maintaining this culture if you see same number of colonies in both non-selective plate and selective plates and start up with new set of electroporation.

[rameshcdri]

Thanks Vete and Kumar

Selected plate  colonies  were not very nice as it should be as usual. Then I discard all the plate and doing electroporation again. I will update you if I will get colonies.

Thanks again.

Ram