Protocol Online logo
Top : Forum Archives: : Molecular Cloning

How to induce self-ligation of a vector? Self-Circularization? - (Jun/03/2008 )

Pages: Previous 1 2 

Try to transform bacteria whith your small account of religated plasmid and innoculate it for making midi or miniprep

-Neko Tonegawa-

^^ Without an ori, transforming won't do anything.

Interesting project!! I don't really understand how it works so can't offer much help to get it done, but maybe we can figure out ways to minimize DNA loss? If I understood correctly, your biggest loss comes after ligation, where only 5% circularized? How do you dialyze? Can you ethanol ppt instead of speed vac'ing?


QUOTE (rosewater @ Jun 11 2008, 12:21 AM)
Reconstruct your vector such that there are gamma delta resolvase sites flanking your region of interest. Treat with with gamma delta resolvase in vitro. This will give you singly linked catenanes. Linearize the circular molecule that you don't want, then gel purify your circular plasmid, or transfect the mixture. Check out Science. 1992 May 29;256(5061):1298-303 and papers by NDF Grindley for more details. The resolvase is very efficient, so it may be worth the trouble. You will have to get the enzyme from a lab that studies it, I doubt it is sold.

--> Wow, thanks for your answer. Sounds promising and I´ll try to religate by this method.


Pages: Previous 1 2