4 replies to this topic

[mobo]

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!

[mobo]

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?

[phage434]

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.

[HomeBrew]

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...

[mobo]

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