3 replies to this topic

[moljul]

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

[BIOKMST]

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 20°C!

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....

[T C]

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 20°C!

[perneseblue]

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.