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Electroporation in Pseudomonas aeruginosa

Pseudomonas aeruginosa Electroporation

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#1 rameshcdri

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Posted 19 December 2011 - 04:18 PM

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?Posted Image 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?

#2 pito

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Posted 20 December 2011 - 02:45 AM

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.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Biouday

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Posted 20 December 2011 - 03:51 AM

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.

#4 rameshcdri

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Posted 21 December 2011 - 10:48 AM

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





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