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Single colony after site-directed mutagenesis?


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#1 HOYAJM

HOYAJM

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Posted 27 March 2012 - 06:58 AM

My DpnI control had no colonies, so the DpnI should be fine. For 4 different mutations I got only 1-3 colonies per plate. For all of my previous SDM work (using the same protocol) I would get a plate FULL of colonies. Is it likely that they contain the mutation?

I am using XL10 Gold cells, I grow out the transformation mixture for 1h at 37C in 500 uL NYZ broth. Then I plate 150 uL of the mixture on LB+Amp plates

I will be inoculating LB+Amp with the colonies and will see if they grow with antibiotic. But in the meantime I was just curious if people have had the same experience and found that single colonies on a plate after SDM could in fact contain the mutation.

Thanks

Edited by HOYAJM, 27 March 2012 - 07:00 AM.





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