Restriction Enzymes

[fmattei]

Dear all,

I'm new of restriction enzymes. I need to digest mouse genomic DNA with two NEB restriction enzymes (separately): HhaI and McrBC. Although I accurately read their datasheets, It is not clear how many of these enzymes I need to use. I must to digest at least 5 ug of gDNA in a volume of 25 ul. By reading some references I can try with 10 Units of both enzymes for 5 ug of gDNA. What do you think ? Is it the right quantity?

Many tahnks in advance for your help.
Fabrizio.

[CrowSan]

Normally RE's come at a concentration of 10 units/ul. A unit is the amount of enzyme required to digest 1 ug of DNA in 1 hour. So 1ul (10 u) theoretically should digest 5 ug in 30 min. However I usualy digest for 4 hours routinely to make sure.
In other words .. Yes. 10 u should be sufficient.

[mchlbrmn]

Yes "theoretically" 1 unit digests 1 ug in 1 hour. However, as is Crowsan's practice, most of us usually use more. In practice sometimes digests don't go to completion, so usually I use 4-10 u/ug and digests as long as is convenient, sometimes overnight (unless I suspect the DNA isn't clean and might be degraded). The McrBC isn't active more than one hour, and hhai rest enzyme for under 8 hours, NEB's site says.