Hi, I have left restriction digested and rSAP treated DNA (originally plasmid of 7Kb, now linear of same length) at room temperature for a week. Will it be alright to use, or rather degraded? (or any other thing)
If you didn't purify the DNA from the enzymes it may be degraded. You may want to check it's integrity on gel electrophoresis. Though, even if you see one band at 7 kbp, the sticky ends should have degraded for sure. You will have much lower ligation efficiency, if any at all.
I agree with Dmitry that the DNA in your reaction volume might be degraded. If you have enough of the DNA you want to cut I wouldn't take any chances and prepare a new restriction reaction.
Cheers, Christian
hi Lobregat,
I hope it will be stable if digested product of DNA is there as it is.But if your product is added along with the restriction enzymes then degradation will be possible.Any way once you try to resolve 200ng DNA on 1 %agarose gel and check if degradation is happened or not ....
all the best
hi thanks for all the feedback, in the end I redid everything and the colony PCRs were promising.. Thanks anyway!
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