RNA-gel; how much RNA can I use? - (Jan/24/2006 )
I've done a northern blot trying to find some transcripts being expressed at only low levels. I used 15-20 µg of total RNA and got very weak signals (with, for some of the probes, somewhat too much background... - I'm using 32P, by the way...).
Now I want to try again but with significantly higher amounts of RNA. I could of course isolate mRNA, but then I'll have to buy some magnetic beads or something and I can't wait for that (I have some oligo-d(T) beads already but they expired in october 2001... time flies...) . So, I wonder if anybody knows how much RNA can I put in my wells before overloading the gel? Good, old Sambrook, Fritsch and Maniatis says max 30 µg/well, but I'm hoping that someone has tested higher amounts...? I use a 1 % gel with formaldehyde.
i did use 50µg and saw no particular pb of migration
That's good news .
How did you run your gel? Low voltage over night with high levels of formaldehyde, or somewhere around 100 volts for about 3 hours with more moderate levels of formaldehyde?
it was on both acrylamide 15% (at 180V) and formaldehyde/agarose gel (80V) (8,75ml formaldehyde 37% in 150ml final gel volume).
Both gels run at RT and for approx 3h