maximum [EDTA] in restriction digests - (Jun/05/2005 )

Just wondering if anyone can tell me what the maximum EDTA concentration is that can be used successfully in a restriction digest. If possible, I'd like to digest 40 ul of plasmid dissolved in TE buffer in a 100 ul (or maybe 200 ul) reaction, which would give a final [EDTA] of 0.4 mM (0.2 mM). I seem to remember using 50% (vol/vol) DNA solution in a past digest, but I can't remember the outcome.

Any suggestions? Or, is there a reference out there somewhere?

Many thanks in advance,

DC

depends on your restriction enzyme.

if it requires divalent cations then you may rune into issues as EDTA chealates them.

However in saying this, I have performed MspI digests on TE resuspended DNA upto half the final restriction enzyme reaction volume with success.

I figure such a high TE concentration could induce star activity, however for MspI at least, this depends on your enzyme.

You could desalt your DNA with a simple G50 column that should remove most of the EDTA in your TE solution no worries.

Good luck

Nick

Thanks for the reply, Nick. We ended up using 10% (v/v) DNA/TE and the digest worked just fine. But I'm pretty sure even 20-25% would be OK.

Cheers, DC