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if you plate 40 ul of your electrocomp cells only... - would you get any colonies? (Jan/11/2007 )

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QUOTE (fred_33 @ Jan 16 2007, 03:23 AM)
Ok. excuse me often i'm unclear.
I mean after electroporation of a plasmid that contain the AmpR gene, i let agitate the bacterias in LB for 30 to 45' in general.
When the plasmid holds the KanR gene, i've compared the time necessary for the bacterias to be ok for plating. I've compared 30' and 1h and noticed that 1h is better than 30' of agitation.

I think it's because ampicilin just block formation of outter membrane of bacterias, which does not kill them but they just stop growing (which explains satellites bacterias which just take advantage of ampicilin degradation by AmpR bacterias). The kanamycin works by affecting 30S ribosomal subunit and causing destabilization of it and a frame-shift. then protein can not be sythetized and the bacteria DIE.
So bacterias seem to need more time before plating after an electroporation with kana.

Fred, thank you for your clarification. I usually let them shake around 1 hour. Now im beginning to think that kana is actually fine but im pouring it into hot media unsure.gif . Because yesterday i incubated some bacteria in kana medium and they died. so the antibiotics should be fine, but maybe extra heat sensitive dry.gif


QUOTE (Kathy @ Jan 11 2007, 06:16 PM)
I have tried different cells mine and those of my friend and it seems that if i plate them on the plate containing antibiotics, some colonies grow blink.gif . nowadays, i use kanamycin, so it clearly depends on its concentration. more antibiotic, less colonies. i even used another method: cool and make agar plates with no antibiotics. then spread 40ul of kanamycin on the top. let dry and spread e-coli. colonies only grow at the edges of the plate. so it is clear to me that it depends on the antibiotic. also plates that i cool more (to the degree that they are all waves when dried rolleyes.gif ) i get less colonies. what do you think? bad antibiotics or usual case?
and plaese can you tell me where do those colonies come from??? sad.gif i autoclave everything. work near fire, dont breathe.... glare.gif why those colonies?????


If i understood you correctly, there is difference between your and your collegues's antibiotic stock and consequently the amount of colonies you will find on your plate. Did you check what kind of strain your collegue is using? From strain to strain and especially in E.coli you will find difference in antibiotic resistance.The strain you are using might need higher concentration of antibiotic to prevent growth. The simplest way to check what would be appropriate amount of antibioitc for the stain you are using is to do MIC testing (minimal inhibitory concentration).


Also, are the colonies that grow E. coli? Do they look like E. coli? In any case, is your colleague using from the same antibiotic stock? If your colleague is having success using the same strain and the same the antibiotic stock then you must have a contamination occurring somewhere along the way.


thank you for your replies, they are so useful. i guess the antibiotic stock is the same, but no the strain is different. MIC is very useful hint thank you i'll try that. But i dont know if they look like E-coli. After around 18 hours of incubation they are large comparing to normal E-coli blink.gif . My freind here tells me that it is first time she sees such large colonies on the plate overnight. they definitively bigger than those of the electroporated with plasmid wacko.gif .


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