4 replies to this topic

[jessh]

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?
Thanks!

[swanny]

jessh, on Mar 11 2009, 10:31 AM, said:

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?
Thanks!

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.

[Nrelo]

jessh, on Mar 10 2009, 03:31 PM, said:

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?
Thanks!

commerically available enzymes, particular EcoRI,HindIII etc, are quite robust and show very little star activity under routine digestion conditions.

[microgirl]

jessh, on Mar 10 2009, 04:31 PM, said:

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?
Thanks!

I routinely digest things overnight. As long as it's not an enzyme that generally shows star activity you should be fine.

[T C]

Hey

Its okay........I also go for overnight digestions.

Best
TC