plate problem--minimal colonies - (Oct/02/2006 )

hello again,

I wrote earlier that i had made plates using LB and amp and they had bubbles. I transformed my bug again (as it was over a week) on the same plates streaked with more amp . i grew them again, this time on three plates i see either huge colonies or very tiny ones, but not the hundreds of colonies i am used to seeing.

i am going ahead and inoculating them overnight, but what went wrong?

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without knowing how much amp was added, I would point my finger at the Amp. How much was added? And how good/even is your streaking?

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all i did was add 20uls or so of the usual amp conc we use. i think my streaking is okay--how would i know that it is not? i did get colonies.

i followed my transformation protocol to the T, dont think that was a problem.

any suggestions?

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My idea was that the Amp concentration when through the roof with the extra Amp. Either killing the cells before they can produce sufficient B-lactamase or by saturating the enzyme. What does 20ul translate too? So in total how much Amp is on the plate? As for streaking powerness... do you cells look uniformly blue when using X-gal/IPTG colour testing?

Anyway, you did get colonies. And as my labmate is fond of saying, "You only need one."

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ha ha nice quote. we dont stain in my lab. is that bad?

yr prob right about the amp, but i was worried there was not enough amp on my plates to begin with (because they were over a week old) so thats what i did.

thanks tho...

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Well I would also hav added xtra amp if using old plates( which i rarely do).
If amp conc is less, sm untransformes may grow (maybe those r small colonies).
Just MY hypothesis.

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