DIG Northern - faint bands and high background (Jan/28/2005 )
I'm going to try Northern blotting on total apple RNA using non radioactive probes (dsDNA DIG labeled probes). How can I bypass the main problems (faint bands and high background)?
i've never done a DIG northern but i have lots of experience with radioactive ones (32-p labelled probes).
if background is a problem then there are several things you can do. first thing is look at the composition of your hybridisation and pre-hybridisation buffer. does it have blocking agents? such as sheared salmon/fish sperm DNA, and/or yeast tRNA, and/or Cot1 DNA?
Also, the annealing temp is important. often increasing the annelaing temp will decrease your background, but if you go too high then you may not get any annealing fo the probe. next thing to change is the washing temperature. the higher you go the less background (and signal if not careful) you will get.
does the DIG labelling protocol include a probe purification step? or do you add it directly to your northern after you label it? i would recommend an ethanol precipitation (with sodium acetate) to get rid of excess free nucleotides. although DO NOT do a phenol:chloroform step, as the DIG will separate into the non-aqueous phase, taking your probe with it! the cleaner your probe the better the hybridisation and signal.
you can also try pre-hybridising for longer to prepare your membrane and block any non-specific annealing.
and lastly, the cleaner the RNA prior to binding it to the membrane, the better the signal you will get. and i guess the amount of RNA you run on the gel will affect the signal. i work with animal tissue, but i normally run 10 to 15 microgrammes of total RNA for each sample.
I'm going to try and your suggestion will help me very much