if you plate 40 ul of your electrocomp cells only... - would you get any colonies? (Jan/11/2007 )
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 . 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 ) 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??? i autoclave everything. work near fire, dont breathe.... why those colonies?????
ok, 2 options. 1, the antibiotics are going off. 2, you've got plasmid contamination in your e.coli stock.
with kanamycin, i used 25ug/mL, put it in the cooling agar, just before pouring the plates. if you put the antibiotic just on the top, you'll never cover the entire plate, and you'll have the border of colonies growing which you've seen.
i'm not sure i understand the last part. you're working with bacteria... that's where the colonies are coming from. or you could just check things out by placing an agar plate with antibiotics and no bacteria in the incbuator, and see if colonies grow.
vetticus, thank you for your suggestions. I did that and no, no colonies grow on the empty (antibiotic plate). I think the antibiotics are going off, though noone in the lab is ready to admit it . about the bacteria part i meant that for example, if i take stock that my freind did only 4 colonies grow on the antibiotic plate, but the stock that i did >50 grow. and it is always like that, stocks prepared by me ALWAYS have some colonies growing on the antibiotic plate.
and no it is not plasmid contamination, i checked them, they have no plasmid.
are you adding anitbiotic to hot agar? You need to cool agar to 45-50 degrees before you add the antibiotic otherwise it will degrade at the higher temperature losing acitivity.
yes i cool. i dont know maybe they have become super sensitive to heat? i cool till i can keep my hand on the glass.
you just take an alliquot of bacteria and smear it on an antibiotic plate, and you get colonies??
are you absolutely sure it isn't plasmid contamination?
if not, you're antibiotics are probably dead (sorry).
or perhaps they've become super bugs resistant to antibiotics??
exactly. i am thinking why comparing between the cells and I made and my freind made there is difference in number of colonies (very few vs very many) but there are ALWAYS colonies. I suspect it had something to do with antibiotics. i hope somebody else starts to work in cloning in this lab so that they start thinking about ordering a new batch
hem well kanamycin is more strong than ampicilin and in my hands, let the bacterias recover for 1h is far better than 30' only... not too sure if that's really relevant but may help.
Fred, sorry i didnt understand what you mean.
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.