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restriction digestion - how long are restriction overhangs intact?? (Jul/30/2009 )

want to know how long fragments with overhangs are intact for ligations? did my restriction 2 weeks ago, and stored products at 20C!

-moljul-

moljul on Jul 30 2009, 04:49 AM said:

want to know how long fragments with overhangs are intact for ligations? did my restriction 2 weeks ago, and stored products at 20C!



I have frozen digestions overnight at -20 and ran gels the next day with successful cloning. Maybe if they are frozen they are still OK. But, when in doubt and preventing days of no results - take time and do another hour digestion....

-BIOKMST-

You mean -20. It should be fine. I generally make the digested vector once and use it for months.

Best,
TC

moljul on Jul 30 2009, 06:19 PM said:

want to know how long fragments with overhangs are intact for ligations? did my restriction 2 weeks ago, and stored products at 20C!

-T C-

i successfully ligated restriction digested DNA fragments that were prepared 2 years ago.

Thus I assume that so long as the digested DNA fragment was prepared without contamination from nucleases, kept on ice/cool and when not in use frozen in TE at -20C, the fragment should remained ligatable for a good few years.

-perneseblue-