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If I were to do a blue/white selection of transformed E. coli on LB agar ampicillin plates at room temperature (23⁰C) for about 2 days and 18 hours, will I run into the issue of satellite colonies or any other issues?

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  • $\begingroup$ Why do you do it this way and not use the classical overnight incubation followed by 1-2 hours in the fridge? Works usually pretty fine. $\endgroup$ – Chris Jan 2 '15 at 21:11
  • $\begingroup$ I normally incubate overnight at 37 C, but I was just curious if it's possible to achieve the same results at room temperature over the span of a weekend (when I am unable to come in to my lab and refrigerate the plates). $\endgroup$ – omg Jan 2 '15 at 21:51
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    $\begingroup$ I have never tried this for blue/white selection, I have overexpressed protein a 18°C to have a higher yield of soluble proteins. I think it will not do harm, as the bacteria grow much slower. If you don't see a clear selection, you can still put them into the fridge. $\endgroup$ – Chris Jan 2 '15 at 22:08
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    $\begingroup$ ampicillin degradation is a real issue at room temperature for more than 1 day. YOu will see a ton of satellite colonies and false negatives $\endgroup$ – jwillis0720 Jan 4 '15 at 9:59
  • $\begingroup$ Yes, once I did it (pBR322's fault) and I did see some satellite colonies. $\endgroup$ – Roberto Feb 7 '15 at 3:43
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You will definitely get satellite colonies. Ampicillin breaks down after about a day, especially exposed to light. Kan, keeps pretty well at room temp, but that does you no good :)

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Since b/w screening is a means to selection, you want maximum selection pressure. This will be obtained by incorporating all optimal conditions. The many enzymes of the lac operon are going to function best at their optimal temp.

Why would you do this? If it is because you can't go in the next day it's better to do it when you're available. You're only going to doubt your results when you get them.

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  • $\begingroup$ Blue/White screening is not a selection step (unless you count picking the right colonies as selection). $\endgroup$ – Chris Feb 5 '15 at 8:49
  • $\begingroup$ @chris you are correct it's just a means to selection $\endgroup$ – rhill45 Feb 5 '15 at 16:24

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