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Enzymes in 10 x buffer - (Jan/07/2008 )

Hey guys,

Sometimes when I'm making a mastermix (particularly for restriction digests) I have a situtation where my enzyme is sitting in solution with the 10 x buffer only (i.e. not made up to 1 x buffer with water). My question is would this denature the enzyme in any way? For example, is 10 x the salt concentration going to denature the protein?

Cheers, Rob

-killerkoz17-

QUOTE (killerkoz17 @ Jan 8 2008, 09:05 AM)
Hey guys,

Sometimes when I'm making a mastermix (particularly for restriction digests) I have a situtation where my enzyme is sitting in solution with the 10 x buffer only (i.e. not made up to 1 x buffer with water). My question is would this denature the enzyme in any way? For example, is 10 x the salt concentration going to denature the protein?

Cheers, Rob

Probably not, unless you leave it there for a few hours. If anything does happen, it will probably be a reversible aggregation reaction by "salting out", which will be readily reversed once you dilute the mix.
Easiest way to avoid the problem is to add the enzyme as the final component of the mastermix.

-swanny-

I don't think having the enzyme in 10x buffer will denature it. I guess most people make up the master mix (for restriction digest) with the 10x buffer and then add it to the samples. And anyway you have the enzyme for a short period of time in the master mix so it should not matter.

-scolix-