I transformed 5-alpha E.coli and plated onto freshly made agar plates with ampicillin (which has recently been made by another member of the lab). Less than 24 hours later I checked and my plate had colonies growing (attached photo). I picked 10 colonies into LB broth + ampicillin, placed in bacterial shaker (37 degrees, 220) and next morning, nothing has grown. Spoke to other lab members and they said the colonies look weird as they have only grown in one area of the plate. I'm wondering if some are satellite colonies but I did pick single colonies towards the outside to avoid this. Thoughts and advice?? It also cannot be my LB broth as I have picked colonies from another plate with the same broth and ampicillin and they have grown up perfectly.
I have currently re-transformed fresh bacteria with my plasmid and streaked onto fresh plates but what could be happening??
Hi sara
I think the agar plate was not properly made with improper or uneven ampicillin concentration.
Those do not look like real ampicillin resistant colonies to me but rather just some background colonies on the plate. It may be the stock of ampicillin is getting old or the plates were really wet or something similar.
Hi Michael J. Benedik, is this something you see if the agar plates are wet and why?
You can get weird things when the plates are wet. I think enough media diffuses into the water layer and then cells can grow like in a culture and when it evaporates you have a much higher density of cells where the water was pooled.
Although upon looking again at the plate in the picture, I don't think that is the issue. Wet plate usually looks more like a thick lawn of cells in a patch somewhere.
But the above plate doesn't really look like the colonies are very healthy, and certainly after 24 hours you should have robust healthy colonies if they were really growing well. So I think the amp concentration might have been too low and just permitted some cells to partially grow. Ampicillin has a fairly short half life in solution (weeks but not months) so it is easy to have a stock solution be much lower than the original concentration. Or plates can age out, but you did say these are fresh plates.
Michael J. Benedik I decided to streak bacteria from the plate onto a new freshly made agar plate and the colonies looked more like what you expect. I grew them up in broth + amp and they surprisingly grew and I have just received sequencing results which suggests they had in fact transformed my plasmid, how odd? What do you think could have happened that when I initially picked from the old plate they wouldn't grow in broth but the freshly streaked ones worked?
That is great that you finally got your clone and it is confirmed. It is hard to say what happened but maybe if there was an issue with the plates the colonies were very mixed with real transformants and some non-transformants that grew on that plate. But restreaking selected for only the real ones.
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