My BL21 cells when grown on an Ampicillin agar plate, completely cover the plate in a lawn as fast as 5 hours. They do this regardless of whether I transform them or not. A plate treated with no cells remains barren, as do plates treated with un-transformed JM109 cells or LB media absent cells.
I have tried fresh ampicillin.
Can my BL21 competent cells somehow have gained ampicillin resistance? I can think of no other cause. Yet I can also think of no reason how they might have done so. They were made competent by the calcium chloride method and have been stored at -80.
It seems very unlikely that your BL21 cells should have acquired ampicillin resistance, especially since you did nothing to select an ampicillin-resistent mutant. More likely, your batch of purported "BL21" cells are heavily contaminated by some other bacterium that is resistent to ampicillin. Alternatively, there could have been a misidentification of the strain you are working with. I would redo the preparation of competent cells starting from a stock that is guaranteed to be BL21. As a first measure, I would check that it is sensitive to ampicillin.
It seems very unlikely that your BL21 cells should have acquired ampicillin resistance, especially since you did nothing to select an ampicillin-resistent mutant. More likely, your batch of purported "BL21" cells are heavily contaminated by some other bacterium that is resistent to ampicillin. Alternatively, there could have been a misidentification of the strain you are working with. I would redo the preparation of competent cells starting from a stock that is guaranteed to be BL21. As a first measure, I would check that it is sensitive to ampicillin.
Your plates could also be too old. Here is a site with useful tips and tricks https://bitesizebio.com/10188/whats-the-problem-with-ampicillin-selection/
By fresh ampicillin do you mean you made fresh plates or you made fresh stock?
Do you let them go overnight? Sometimes a heavy inoculum of cells will grow for a while on antibiotic plates before enough of the antibiotic enters the cells to kill them.
Do they grow in a lawn when you dilute them?
You could also try switching to carbenicillin or upping the ampicillin concentration. Otherwise it's probably like Pierre said and you just have the wrong strain or contamination.
Thank you for your responses. Yes the plates and ampicillin were both made fresh.
I started again from fresh BL21 cells and the problem is now fixed. I believe Pierre is correct, either the cells become contaminated with an unknwon cell or I was given a mislabeled cell line.
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