Double Digest - (May/14/2010 )
Hi,
Does this procedure sound correct for a double digest of plasmid DNA using NotI and Xba1?
3 uL of DNA (2 ug)
4 ul NotI enzyme
4 ul XbaI enzyme
5 ul Promega Buffer D 10X (compatible with both enzymes)
Water to 50 ul
Incubate at 37C for 1 hour.
Thanks!
CHurst5841 on May 14 2010, 12:14 PM said:
Does this procedure sound correct for a double digest of plasmid DNA using NotI and Xba1?
3 uL of DNA (2 ug)
4 ul NotI enzyme
4 ul XbaI enzyme
5 ul Promega Buffer D 10X (compatible with both enzymes)
Water to 50 ul
Incubate at 37C for 1 hour.
Thanks!
Never used Promega enzymes, only NEB, but for us, that would be WAY too much enzyme. I rarely use more than 1 ul, even for up to 4-5 ug of DNA.
I agree with fishdoc -- the total amount of restriction enzyme(s) added shouldn't exceed 10% of the final reaction volume -- I usually try to stay a bit under that. The reason is that most enzymes are shipped stored in a buffer containing 50% glycerol (Promega's NotI, for example, is shipped in 10mM Tris-HCl (pH 7.4), 0.1% Triton X-100, 500mM KCl, 0.1mM EDTA, 1mM DTT, 0.5mg/ml BSA, 50% glycerol, while their XbaI comes in 10mM Tris-HCl (pH 7.4), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.5mg/ml BSA, 50% glycerol), thus adding enzyme equivalent to 10% of the final digestion volume makes that digestion 5% with respect to glycerol.
Glycerol concentrations higher than 5% inhibit most enzymes, thus you want to stay below that amount.