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Ideal incubation time


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#1 Ethereal

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Posted 17 March 2009 - 04:34 AM

Hi ,

Can someone enlighten me on why the incubation time for ampicillin plates should not be more than 16 hours?

I have been told, ideally, incubation time should be about 12-16 hours, but why when it passes the 16th hour mark, it is not ideal anymore?

#2 bob1

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Posted 17 March 2009 - 03:39 PM

This is a bit of a fallacy, they will keep just fine for 48 hours or more at 37 degrees. Apparently there may be some degradation of the plasmid levels if you incubate for so long that the bacteria are out of log phase growth, however, I have never had a real problem with it.

#3 perneseblue

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Posted 17 March 2009 - 05:38 PM

The primary cause is ampicilin degradation. Beta-lactamases is secreted into the agar surrounding the colony on the plate. This causes the ampicilin to degrade. Once selection is lost, you start seeing the emergence of satellite colonies which can obscure the real colony (by growing over it and around it) and can grow large enough to make it hard to determine which is the real colony.

If you are doing colony PCR, old colonies don't amplify well. Once the colony becomes too big, the cells become stressed and start to increase production of lipopolysaccarides. Which seem to interfere with the PCR reaction.

You can grow your colonies longer than 16hr if you drop the incubation temperature.
May your PCR products be long, your protocols short and your boss on holiday

#4 Ethereal

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Posted 18 March 2009 - 04:08 AM

Thank you very much!
=)




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