how long for a double digestion - (May/02/2011 )
I am digestion some plasmids with XhoI and KpnI, how long should that take?is 4h enough or does it need to be overnight? I need them completely digested, they are about 6kb
depends on how much conc plasmid/enzyme you are using, i usually digest 1.5 microgram of plamid with 2 microlitres of each enzyme (NEB) for 3.5 to 4 hrs.....it works fine for downstream ligation.....
Complete digestion is very difficult in any circumstance. Normally, 1/2 hour digestion is more than enough for ligation. If you are trying to reduce vector background, then I'd recommend amplifying your vector with PCR and digesting the template with DpnI. This reduces the amount of intact circular vector substantially and cuts most of the background.