I made ampicillin plates (1:1000 ampicillin dilution that is 400ul in 4 mls of LB broth) and grew my transformants (dh5 alpha + vector pT7-FLAG2 + DNA inserts)into the agar overnight at 37 celsius and the next morning they are growing all over the plate! I did this before and there was no problem. I got nice single colonies. But I repeat this procedure twice and kept getting a carpet-like blob of colonies every time I grow them on the plate.
I think perhaps, the competent cells can be contamined or the antibiotic may be corrupted.
I hope you also had a control plate. Sometimes it is just not about having a carpet of colonies on your plate where you spread your cells carrying your ligated sample. You can draw conclusions when you compare it with a plate grown with cells having just your vector without insert. And also check if you had dephosphorylated your vector before setting your ligations.
The above mentioned answers cover most of the cases that be the problem. Also you could try to use higher concentrations of ampicillin in LB so as to see how resistant your cells are.
The three most likely causes, in my experience:
- You added Amp to the molten agar while still too hot.
- Ampicillin stock too old.
- Plates older than 1.5-2 weeks.
- Competent cell stock prepared from AmpR strain.
Always include a non-transformed control in your transformations. If that also yields a carpet of colonies, check for one of the four possibilities above. Otherwise, you are probably using too much DNA in your tranformation.
Along with all above points also check the number of cells (cell-count) you are using for the experiment.
Alright. Thanks everyone for the suggestions! My colonies are up and running!
how much volume are you plating. Some times you may have to plate multiple volume. Plus follow things about the sterility, control plates etc etc.
First of all, I am confused by your dilution (400ul into 4ml?). secondly, are you using fresh plates? In that regard as well, are the plates being stored properly? Ampicillin plates left out overnight on a bench at RT should not be used. In my experience, long periods at RT or higher temps degrades the antibiotic activity.
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