I am having problems with Northern blots. After the transfer I have quite little RNA on my membrane and quite a lot still in the gel (RNA quality OK) and the 28S rRNA is only very weak/not transferred. (visualized by Methblue staining) The Northerns give very weak signals after hybridisation even for highly abundant genes.
Can somebody help me with this problem? Has somebody else the same problems?
What I am doing: Run a 1% formaldehyde/agarose gel, wash the gel in water, then denature in NaOH and neutralize in Tris (skipping the last 2 steps doesn't make any difference) and finally shake the gel in 20XSSC. Prewetting the membrane in water, and 20XSSC. set up the transfer in 20XSSC (capillary) and transfer overnight, followed by UV-crosslinking
Thank you very much,