how do you inactivate the enzymes after digestion? - (Nov/28/2005 )
i wonder what method all of you here used to inactivate the enzymes.my PCR product was digested by EcoR I and BamH I,now i wanna inactivate the enzymes before ligation.any suggestion is appreciated.
I always gel purify my digested PCR product anyway, and thus have no need to inactivate the enzymes. Aren't you going to run your digested PCR product out on a gel?
nope! coz i have many many samples.if that,it will take a lot of time and be boring,any good method?thank you though.
standard procedure to inactivate enzyme is 65° for 20'.
To be sure, i do 30'.
But bamHI is not sensitive to heat inactivation as said by NEB website...
Firstly,i add 2X 100% ethanol to precipitate the digested PCR products,and dry them by vacuumizing. is that ok?
heat drying would be less harmful for your fragments.
after your 100% etoh, wash them in 70%ethanol to remove salts
Heat inactivation is the best way to go but as pointed out by fred BamHI is heat resistant. For future reference, NEB has a nice table that summarize the temperature required for each enzyme.
For heat resistant r.e., I usually do a phenol:chloroform extraction follow by a chloroform extraction. This usually inactivated the restriction enzyme. For this procedure, you might require higher volume (min of 40 to 50 uL). If your volume is too low you can raise it with TE or water, do the extraction then EtOH precipitate.
would an ETOH precipitation of an RE digestion be sufficient to get rid of the REs?
I was wondering that the other day, but assumed it was probably not sufficient....I think ETOH ppt is mostly about getting rid of excess salt and small contaminants, or to concentrate the sample, but not really for getting rid of proteins...
what do you think? has anyone here ever tried to do it like this: RE--> etoh ppt--> ligation rxn
as i do double digests, i gel purify my dna so i assume i get rid of enzymes
that's usually how I do it; I was just wondering if it were possible to save the time ...but, as we're only talking a few hours it's not significant....I was just curious mostly