Protocol Online logo
Top : Forum Archives: : Molecular Cloning

XbaI/BamHI double digest- dam methylation detection problem - (May/08/2006 )

Hi all!

Aim: double digest a plasmid with XbaI and BamH I propagated in E.coli (dam methylase positive) to cut out the insert.

Results: after various conditions tested, conclusion is that XbaI doesn't cut it. buffer ok, other conditions ok.

Question: XbaI is sensitive to dam methylation. How can I test if Xba I site on this plasmid has dam methylation on it? Is the only solution to propagate it in dam minus E.coli strain? (I hope not!).

If needed, for better understanding, I can provide more details.

Thank you to everyone who tries to help me, I am now really stuck with this.

GK cool.gif

-GK Sydney-

as i know, in a classical E. coli, all the dam sites are methylated. so JM110 strains is the most suitable for your exps.
If you don't want to do jm110, you have to PCR your whole vector (pcr does not methylate laugh.gif)


Yes, I know, the one I am using is MC1061/P3 and it deffinitely methylates it.

I am trying to find out how I can check if it is methylation that blocks XbaI from cutting....if not, then I should probably sequence to check for mutations at the site....

any further suggestions?

thanks cool.gif

-GK Sydney-

well you can try to digest vector by an other dam sensitive enzyme and maybe see the products....