does anyone know if it is really necessary to purify a radioactive DNA probe
after labelling it by random priming? I want to use it on a northern blot.
So far, I worked with DIG-labelled probes, that do not need to be purified
after labelling. They are used directly in the hybridisation solution. Is it
different with radioactive probes? Do you think that precipitation by salt
and alcohol is sufficient for purification?
"Simone Marker" <[Only registered users see links. ]-kl.de> wrote in message
news:faui8f$ltc$[Only registered users see links. ]-kl.de...
I *never* purify my probes (southern or northern). I label, and mix directly
with the hyb solution. It's not inconceivable that you may get a slightly
higher background, as some people claim, but I haven't noticed background
problems in many years, so I'm sticking with no-purification. Of course, if
you want to check that your labelling reaction worked, you still need to
purify it (or check a spot with TCA), but having a bad labelling reaction by
random priming is such a rare event, that I don't bother checking either.
We purify our radioactive probes using small gel filtration spin columns
(I think it's called MobiSpin) - you get rid of most unincooperated
radioactivity, which is retained in the column, while your labelled
probe is in the flow-through. It makes it much nicer to work with the
probe because it is not as radiactive as the whole reaction mixture..