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transformed bacteria doesnt grow - (Aug/24/2010 )

Hello,

I obtained a plasmid mailed to us from filter paper and transformed it into DH10B. I saw colonies in ampicillin plates and picked up several colonies to grow in 4 ml LB broth to make maxipreparation of plasmid. However, when i tried to incubate it for 12 hours on the broth, it doesn't grow. I dont know the problem since, there were colonies that formed from the plates. what should i do?

thank you

-soymilk14-

soymilk14 on Wed Aug 25 05:31:33 2010 said:


Hello,

I obtained a plasmid mailed to us from filter paper and transformed it into DH10B. I saw colonies in ampicillin plates and picked up several colonies to grow in 4 ml LB broth to make maxipreparation of plasmid. However, when i tried to incubate it for 12 hours on the broth, it doesn't grow. I dont know the problem since, there were colonies that formed from the plates. what should i do?

thank you


Take that colony and re-plate it on a fresh new Ampicilin plate.

There are 2 possibilities for the situation you have just described.
1-What you are seeing are real plasmid carrying colonies. If so the plasmid you have is a single copy BAC, and your e coli strain grows slowly. How long did it take for the colonies to form on the Amp plate? Did you use a fresh Amp plate. WHat is the concentration of Amp used? Ampicilin can nturally hydrolyse
.
2-What you are seeing are false positives. Either the Amp plates are old or you have incubate the plate for so long that the amp lost its activity.

-perneseblue-