4 replies to this topic

[Samantha]

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 ?

thanks

samantha

[pcrman]

I am curious how you know the digestion is not complete?

[Samantha]

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.

[peixumol]

try to further purify your DNA,because very few  pollutants may inhibit the activity of  the  restriction enzyme.

[chandima]

hi
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.
chandima