restriction digest and DNA degradation - (Oct/10/2005 )
For the past week I have noticed that my DNA gets badly degraded after restriction digest. I think that as a consequence, my subsequent ligation step is poor . Has anyone had that problem? how did you deal with that? please I'll be waiting for your advise
No, I think you are doing something wrong. For instance some restriction endonuclease preps will exhibit star activity at certain salt concentrations (mostly from an impure enzyme prep.) and cause secondary cutting and other sites. DNA should be stable in all the enzyme buffers however unless you add DNase.
I agree with tap14 -- something's wrong. Can you give us a bit more info on your experiment and it's results? What if you do a "no enzyme digest" -- set up the restriction digest the same way you always do -- DNA, water, 10x buffer -- but just add more water (to compensate for the volume of enzyme you would have added).
Incubate this as you would for a restriction digest (perhaps along with a real digest done at the same time using the same materials), and run a gel with lanes of undigested DNA, your no-enzyme digest, and your real digest.
check other things also like TAE buffer, agarose, water etc !