Double digest with EcoR1 - would it work? - (Dec/14/2004 )

Is it worthwhile to digest twice with the same enzyme?

I have digested genomic bacterial DNA with EcoR1 but the fragments are still quite large (after 4hr at 37 degrees).

If I take some of the digest product and re-digest with the same enzyme is it likely to cut any further?

Do I need to inactivate the first digest or can I just add more enzyme (and would I need to also add more buffer or BSA?)?

Thanks

EcoR I is subject to star activity if there is too much enzyme/DNA concentration/volume. Excessive digestion also increases the risk of exonuclease nibbling of the ends.

In my hands, 80 units of restriction enzyme in a 150 µL restriction reaction for 3.25 hours at the recommended temperature are required to completely digest 25 µg of pUC vector (equivalent to 14.4 pmol). Since 25 ug of pUC = 8.7 e 12 molecules, each of which contain one EcoR I site, 80 units of enzyme will safely cut 8.7 e 12 EcoR I sites in 3.25 hours without generating star activity. This rule varies according to the unit definition of the enzyme, as some are defined on linear DNA while others use supercoiled plasmid. Some enzymes are not as efficient on superecoiled plasmid DNA. (See the New England Biolabs catalog.)