partial digestion of Tth111I - difficult??? (May/08/2007 )
have you ever try to do partial digestion of Tth111I??? for construction of my expression cassete may be i need to do this cause i failed to find any other suitable enzyme site.
i cannot decide how to digest it partially cause within the same insert fragment there is 3 sites and also i am confused how to inactivate the enzyme
thanks in advance for your help
I have never tried using an enzyme as exotic as Tth111I. However after reading about this enzyme's characteristics on NEB's site, I would strongly advice not to use this enzyme.
I would suggest trying Tth111I isoschizomers, (enzymes which recognise the same restriction site) like PsyI (fermentas) and if failing that PflFI (NEB). Both have superior characteristics, namely they can be heat inactivated (useful for partial digest), have superior cutting ability - the ends they produce are more likely to religate, and have far lower star activity. And most important of all cheaper
Enzymes can be inactivated by heat, and removed by phenol/chloroform extraction. A final concentration of 20mM of EDTA is usually good enough to remove the Mg ions, temporary stopping the enzymes from working.
So what i do, is add EDTA when I want to stop the reaction. Phenol/chloroform to remove the enzyme. Then I heat kill the mix. (for superstitious reasons I don't heat skill prior to the phenol/chloroform step.)
My prefered method for partial digest is to make a series of enzyme dilution , 1:1, 1:5, 1:10, 1:20, 1:50, then use the same amount of DNA (15ug), incubate the mix for 1hr. Check an aliquote and flash freeze the rest on dry ice. Fine out which vials contains the most linear DNA. If not enough cutting, quickly reheat tube on heating block and incubate for ~30min.. repeat
Once correct vial identified, EDTA, phenol/chloroform,heat and gel purify desired linear fragment from uncut DNA.
This method is certainly very wasteful with DNA, but as I roll in buckets of plasmid DNA, I am not concerned.
thanks for your advice and info. i didnt find any other way but use that enzyme.
i was also panning to do serial dilution to find out the best reaction condition.