Accidentally digested DNA overnight - still OK? - (Mar/10/2009 )
I accidentally left my EcoRI digest overnight @ 37 degrees (~17 hours). I used 1.7 ug 6.5 kb plasmid DNA and 10 U of enzyme. Will the plasmid still be OK for used in QPCR?
jessh on Mar 11 2009, 10:31 AM said:
Run it out on a gel and see. There is no reason the DNA will be degraded (unless you added too much enzyme solution - greater than 10% of the reaction volume - thus raising the risk of star activity), as an overnight digestion is a standard part of the QA for the enzyme.
Why not try a QPCR and see? You'll never know unless you give it a go.
jessh on Mar 10 2009, 03:31 PM said:
commerically available enzymes, particular EcoRI,HindIII etc, are quite robust and show very little star activity under routine digestion conditions.
jessh on Mar 10 2009, 04:31 PM said:
I routinely digest things overnight. As long as it's not an enzyme that generally shows star activity you should be fine.
Its okay........I also go for overnight digestions.