Last month I stored a double-digested vector backbone after cleanup/purification at -20 degrees and did ligation with my insert but got a self-ligated product. Is it possible that its overhangs get degraded?
DNA is quite stable (even more if purified and stored bellow 20°C.
did you do a DNA fragment gel purification or just a purification of the whole digest?
depending on your type of purification you did you may think;
1-your double digest is not 100% OK (some sites are not opened and it is easy for the ligase to close the circle)
2- the two fragments have been religated and the initial plasmid reformed
...
you may also check whether the restriction sites you used could be compatible ...
There are a couple of controls you can run to try and figure out how much uncut or cut and self-ligated plasmid you got. Here is an example:'
tube 1: cut and purified vector, ligase buffer, water, NO insert, NO ligase
tube 2: cut and purified vector, ligase buffer, water, NO insert, with ligase
tube 3: cut and purified vector, ligase buffer, water, your insert, with ligase
tube 1 will let you know how much vector was uncut
tube 2 will let you know how much vector was uncut or was single cut and self-ligated
tube 3 is your actual ligation reaction.
Ideally, tube 1 and 2 have 0 to very few colonies, and tube 3 has more.
Yes you do get some degradation of the ends which results in blunt end ligations. Generally only store cut DNA for about a week.
In theory it should be ok for a while longer. Ideally, it's best to cut and ligate with fresh DNA so you don't get degradation.
If your cloning is going well with frozen DNA then it's fine to use the frozen pre-cut DNA. However, if cloning is not going well, then switch to only freshly cut DNA is a good option.
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