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Topic: Plasmid cloning  (Read 15390 times)

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Offline madscientist

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Re: Plasmid cloning
« Reply #15 on: March 10, 2007, 12:31:05 AM »
I cant get my head around why there is a need for there to be an intact functional antibiotic resistance gene in the recombinant plasmid in order to select colonies containing the recombinant plasmid? I know I must be missin somthing easy but its doing my head in  :-\
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Offline Yggdrasil

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Re: Plasmid cloning
« Reply #16 on: March 10, 2007, 12:48:37 AM »
After you ligate your fragments together, you transform the DNA into bacteria, plate the bacteria onto growth media, and wait for colonies to grow.  In the transformation step, not all of the bacteria in the sample take up DNA.  In fact, only a small percentage take up DNA.  Therefore, when you plate cells onto a agar without any antibiotics, you will get a lawn of bacteria, most of which have no plasmid at all.  This is the main reason why you need antibiotic selection: to be able to distinguish bacteria with a plasmid from bacteria without a plasmid.

Offline madscientist

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Re: Plasmid cloning
« Reply #17 on: March 10, 2007, 01:01:29 AM »
so the method shown in the pic above (replica plating) wouldnt work? wouldnt you be able to tell the plasmids that have taken up the DNA from those that havnt by seeing which ones will grow on the plate without antibiotic, but die on either tet or amp containing plates?

Quote
Therefore, when you plate cells onto a agar without any antibiotics, you will get a lawn of bacteria
Is it becuase of this "lawn of bacteria" that replica plating would not be possible, i.e. too many colonies too close together....? 
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Offline Yggdrasil

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Re: Plasmid cloning
« Reply #18 on: March 10, 2007, 04:08:03 AM »
Any bacteria, whether they carry plasmid or not, will grow on the plate without antibiotic.  These bacteria will also die on either tet or amp plates.  So, you will not be able to separate bacteria containing your recombinant plasmid and bacteria that have no plasmid at all.

Offline madscientist

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Re: Plasmid cloning
« Reply #19 on: March 10, 2007, 08:24:08 PM »
I thought all bacteria had plasmids in them, I didn't realise that after transformation there would be some bacteria that have taken up the recombinant plasmid and some with no plasmid at all.

Thanks again for all your help on this question Yggdrasil your a legend!, much appreciated.

Cheers,

madscientist
The only stupid question is a question not asked.

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