RNA-gel; how much RNA can I use? - (Jan/24/2006 )
Hi,
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.
Regards,
Sonja
i did use 50µg and saw no particular pb of migration
Hi, Fred
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?
Sonja
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