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How to extract a plasmid in a strain carrying two types of plasmids


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4 replies to this topic

#1 mobo

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Posted 18 March 2011 - 06:25 PM

Hi guys,

I received from another lab a cell strain that carries two types of plasmids (with different ori and different resistance markers).
plasmid 1 (ColE1 + Ampicillin), and
plasmid 2 (p15A + Chloramphenicol)

Now I need extract one of the two plasmids (ColE1+Amp) without contamination from the other.

Does any of you know how to culture the cells to get a strain that will contain only one type of plasmid?

Thank you very much!

#2 mobo

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Posted 18 March 2011 - 06:28 PM

I'm currently thinking of the following approach for :

1. Culture the strain with both antibiotics (Amp+Cam)
2. Miniprep the culture to get DNA mix from both types of plasmids
3. A transformation with the DNA mix, and plate onto Agar plate with Ampicillin (without Chloramphenicol)
4. Pick single colonies (say 10 colonies) and streak onto new Amp plates.
5. Inoculate half of each patch into a new tube with LB medium and Chloramphenicol, and grow over night..
6. If I see culture growing: This mean that cell patch contains both plasmids ===> Discard
If I doesn't see cell grow: This mean that this cell patch contains only Amp+ColE1 plasmid. ==> Save and miniprep.


Would take work? This sounds very tedious. Is there any better solution?

#3 phage434

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Posted 18 March 2011 - 08:26 PM

That's pretty much what I would do. You could save some trouble by replica plating the amp plate onto a Cm plate to identify the double transformants, but it would take another day.

#4 HomeBrew

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Posted 19 March 2011 - 03:49 AM

If the plasmids are of different sizes, you could linearize them, separate them by gel electrophoresis, recover the band you want from the gel, ligate and transform...

#5 mobo

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Posted 20 March 2011 - 04:16 PM

Hi Phage434 and HomeBrew, Thank you both for kind replies. It's very helpful to me!




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