1 reply to this topic

[soymilk14]

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

[perneseblue]

soymilk14, on 24 August 2010 - 09:31 PM, 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.