4 replies to this topic

[Ceremony6]

Hi,

I transformed New England Biolabs High Efficiency E. Coli cells with a pBLUESCRIPT vector and my 1.2 kb insert. I got colonies that were 4x more abundant on the experimental LB-AMP plates than the control ligation plates, so it seems like some of the cells should have my circularized construct. But when I inoculated 10 colonies off the plate into liquid LB-AMP, none of the cells grew. To check if there was something wrong with the liquid media, I inoculated my media with E. Coli that was not from my plates, and it grew normally. The only culprits I can think of is that my cells did not take up the amp resistance vector, but grew anyway because ampicillan levels were not high enough on my plates, or that there is something wrong with the cells themselves. I also made sure to avoid picking satellite colonies.

Does anyone have any other ideas as to what could be going on?


I see that another poster is experiencing a similar problem.

Edited by Ceremony6, 02 February 2011 - 01:07 PM.

[phage434]

How long did you grow the original plates?  Colonies appearing after more than overnight are often not amp resistant.  Why did your untransformed cells grow in medium that was supposed to contain antibiotic?  How did you pick the colonies?  A hot loop will kill cells, for example.

[Ceremony6]

phage434, on 02 February 2011 - 01:26 PM, said:

How long did you grow the original plates?  Colonies appearing after more than overnight are often not amp resistant.  Why did your untransformed cells grow in medium that was supposed to contain antibiotic?  How did you pick the colonies?  A hot loop will kill cells, for example.

I grew them overnight.

I don't know for sure if there were untransformed cells growing on my plates, but if there were it might be because the ampicillan was no good, or I didn't put in enough in. I would like to test the effectiveness of the plates with a plain untransformed E. coli strain, but all we have are the really expensive cells, and I don't want to waste them.

I picked my colonies using a sterile plastic loop and swirled it in the liquid medium.

Maybe I should just make new plates.

[phage434]

Your controls are just as important (and are part of) your experiment.  You need to know if your plates inhibit growth of your untransformed cells.  You can likely use a loop to streak a small amount of the still frozen competent cells on a plate and still use it for your next transformation, but even if you could not, you should get into the habit of treating the controls as vital to your experiment.

Did you wait for the agar to cool before adding ampicillin?  High temperatures will decompose it -- you need to cool the agar to around 55C before adding the antibiotics.

[seanspotatobusiness]

Would tight caps on the liquid culture tubes prevent growth?