Genomic DNA Complete Digestion - (Jan/24/2005 )
i digested my genomic dna (10ug) with a restrcition enzyme (100u) twice overnight, together with 0.1mg/ml BSA in a 50ul reaction. however, the digestion is not complete. the OD260/OD280 ratio is 2.
what can i do to make the digestion complete? what do you guys usally do for complete digestion of genomic dna ?
I am curious how you know the digestion is not complete?
i know the digestion was incomplete because I also set up a negative control. There was a very sharp thick band with a large size...which also appeared in the negative control. If the digestion was complete, the thick sharp band shouldnt appear and a smear should be seen instead. Actually, my genomic DNA was digsetd with HpaII which cleaves unmethylated CpG sites, so a smear was expected.
try to further purify your DNA,because very few pollutants may inhibit the activity of the restriction enzyme.
purity is pretty important, do organic extractions to the sample or u can purify it with commmertially available colums. i usually do twise, the organic extraction during DNA extraction.