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wisdom10's picture

Hi All,

I have done transformation and got some colonies on LB plate with ampicilli, but after the miniprep I did the PCR wit my T7 primers and got no result. I've done several times and got nothing. Can someone please tell me what could be the problem?


mnmolecular's picture
Perhaps there is no insert in

Perhaps there is no insert in the plasmid of the colonies you see, therefore no PCR product. 

If you CIP your plasmid backbone after digestion you have less chance of colonies that result from backbone  that is singly cut and then just reanneals.

Also minimize the amount of plasmid backbone DNA and add 2-3 fold insert in your ligation.

You should get may colonies, not just a few and a good ligation should have over 75% that contain the insert ligated into your backbone.

Anything else you can tell us about your backbone, the digestion used to open the backbone and the insert you want to ligate in?