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Accidentally digested DNA overnight - still OK?


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4 replies to this topic

#1 jessh

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Posted 10 March 2009 - 03:31 PM

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!

#2 swanny

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Posted 10 March 2009 - 05:05 PM

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.
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#3 Nrelo

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Posted 11 March 2009 - 03:42 AM

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.

#4 microgirl

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Posted 11 March 2009 - 07:09 AM

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.

#5 T C

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Posted 12 March 2009 - 03:00 AM

Hey

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

Best
TC




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