trouble with restriction digestion - (Jul/14/2006 )

Hi all! I have some trouble with the restriction digestion of some DNA samples extracted by desalting. The NEB enzymes EcoRI and EagI seem to be inhibited in some cases. I always used desalting for DNA extraction, and had no trouble sofar. The only thing I changed was using higher amount of 3M NaCl in the process. Could this be the cause? What agent can inhibit restriction enzymes? Can anyone suggest a method to 'clean my DNA samples? Thanx!

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well, you can P/C/IAA extract?

or perhaps precipitate your DNA with alchohol/oAc, then do the 70% EtOH wash, then resuspend in a buffer without the salt.

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Thanx! I'll try the precipitation! But will it remove the excess salt? Or are there other methods to purify extracted DNA? These samples are quite important and it would be hard to obtain new bloodsamoples to try the P/C method.... and besides: I never had such trouble with desalted DNA... that's why I'm surprised that suddenly it won't work.

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I have never performed a desalted extraction?

I use lysis - P/C/IAA - ISOH ppt - EtOH ppt for gDNA, I use mini-preps for plasmid DNA. but if you have a purified DNA sample that just needs its salt concentration reduced, I can't see why you couldn't do the EtOH ppt in order to change the buffer

if this is a new problem with your DNA samples, what have you changed about protocol?

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