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Why can you plate directly when having ampicillin?

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Why can you plate directly when having ampicillin?

Postby poobear » Thu Nov 18, 2010 3:26 am

When doing transformation, I hear that after heat shock I should wait 30-60 min before plating. However, when Im using ampicillin Im hearing that I can plate directly without wait time. It has something to do with ampicillin slowing down cell growth instead of killing cells? Or something like that. However I dont really understand it..

Someone care to explain to me? :oops:

Thanks
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Re: Why can you plate directly when having ampicillin?

Postby protold » Thu Nov 18, 2010 6:41 am

Hloa, after heat shock actually the plasmid has enter inside the bacteria and gives it the antibiotic resistance and the presence of the gene to clone (or empty plasmid) , but it is better to give to the cells 1-2 generations to adquire again normal characteristics from the competence condition, before platting but I suposse that this process could happen on the agar medium. For that after heat shock ,people add medium and incube at 37º C during 40-60 min. All people that I know incube after shock whatever was the resistence gene. in the first 20 hours , colonies that appears were that have plasmid. At more longer times when betalactamase brokes ampicillin, satellite colonies will apear around the harboring plasmid ones. Good luck
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Postby JackBean » Thu Nov 18, 2010 6:47 am

http://en.wikipedia.org/wiki/Ampicillin ... _of_action

yes, if the cells do not grow, it actually doesn't matter much, whether there is amicillin or not.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby canalon » Thu Nov 18, 2010 1:54 pm

In short:
Ampicillin is active only during cell division. while many other antibiotics are active right away and prevent other essential function in the cell. But since before it reaches the stage where it can divide the bacteria will have had plenty of time to synthesize proteins, including beta lactamase, it will not be a problem with ampi. On the other hand with other antibiotics you are very likely to kill the bacteria before it even has time to express the antibiotic resistance gene. So the delay is to allow correct expression before trying to poison the sensitive cells.
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Re:

Postby poobear » Thu Nov 18, 2010 3:05 pm

canalon wrote:In short:
Ampicillin is active only during cell division. while many other antibiotics are active right away and prevent other essential function in the cell. But since before it reaches the stage where it can divide the bacteria will have had plenty of time to synthesize proteins, including beta lactamase, it will not be a problem with ampi. On the other hand with other antibiotics you are very likely to kill the bacteria before it even has time to express the antibiotic resistance gene. So the delay is to allow correct expression before trying to poison the sensitive cells.

Thanks for a great answer, I wish everyone gave answers like you.
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