gel purification after restriction digestion - (Nov/10/2005 )
i was wondering if it is still possible to perform a phenol extraction of a restriction digest if i accidently added loading buffer to my sample. is there something else i need to add?
Oh I see now your problem, as I answered before, I would run everything onto a gel.
keep your DNA sample at 4C while you prepare a gel with adequate agarose percentage and adequate well size.
If you load all your restriction digested plasmid in a gel ( to run everything you can combine slots/wells with tape) and then you can easily and safely isolate the DNA from gel with a column.
For a 50ul digest I usually use the following gel:
0.7 or 0.8% agarose gel if it is for plasmid isolation
using a medium gel tray ( I don´t know now the size), I use 2 wells combined with tape (out of a 16 well comb) to load 50ul sample. It usually fits well but if you have doubts about making this double size well, run sample in different wells and isolate DNA from gel for every lane.
After elute DNA from column matrix with as few volume of H2O as possible (15 ul maybe) and combine all the purified aliquots.
if yr question is just about getting the DNA out - yes u can... loading buffer in its nature does not interfere with the DNA quality or the DNA its self.
You can either run the sample onto a gel and then purify or go for phenol chloroform extraction. No problem.