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transformed bacteria doesnt grow


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#1 soymilk14

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Posted 24 August 2010 - 09:31 PM

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

#2 perneseblue

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Posted 25 August 2010 - 08:37 PM

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.
May your PCR products be long, your protocols short and your boss on holiday




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