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blue/white screening


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#1 tomgar

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Posted 26 December 2004 - 02:52 PM

Hi everyone
I perform ligation (probably unsuccesful) and after that blue/white colonies screening, I have always experienced strange phenomenon: I get
few blue colonies and around them zones of white colonies, never white colony in center of blue ones or white ones;sometimes i try to pick some white colonies and grow them in new fresh LB plates with ampicilin but they won't grow! Anyone could explain me that?
Regards
Tom

#2 Canalon

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Posted 28 December 2004 - 07:15 AM

That's a problem with ampicillin screening. Resistance is due to beta-lactamase that are degrading your antibiotic. Hence the concentration of ampicillin is rapidly too low around your resistant colonies to allow selection. And a few surviving competent bacteria without plasmid can thus grow around the resistant colony. They are usually called micro satellites because they form small colonies (delayed start of growth) around a big resistant one.

If your plasmid include other resistant genes, try alternative antibiotics. I'm using Invitrogen TA cloning kit, and selection can be achieved with both ampicillin an Kanamycin. The latter give much cleaner results :D




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