northern blot probe - is it necessary to remove unincorporated nucleotides? (Aug/07/2007 )
greetings all, i am looking to label DNA using Ready-To-Go DNA Labeling Beads (-dCTP) from amersham. i just wanted to sort out if the removal of unincorporated nucleotides prior to hybridization is necessary for a nice northern?
yes... you get a background grey across the membrane if you don't removed the unincoperated nucleotide. This makes it harder to see signals which aren't very strong.
many thanks! do you know a cheap and cheerful way to remove these unincorporated nucleotides?money is quite tight!
Old prptocols mentionned the measurements of radioactivity by precipitable TCA fraction.
Well i don't truly know the TCA method but that could be first approach.
Second, the old Naacetate/ethanol precipitation doesn't make nucleotides pelleting.
I agree with fred_33 about the ethanol precipitation. And you could whistle while you work to make it cheerful.