nucleic acid fixation - how?... (Aug/03/2005 )
basically, manufacturer's recommend to fix nucleic acids by UV. But i got problems. Don't know the time of exposure, and don't know the energy of my UV lamp (but know the wavelenght).
Faing these pb, i was wondering are they alternatives at uv?
I've heared about baking membrane at 80° fo 1 hour. But as i'm interesting in siRNA is it efficient and/or suitable for detection by northern blots?
Thanks in advance.
You can bake but I think only for nitrocellulose membranes and not nylon. Do you want to fix siRNA to the membranes or detect siRNA using DNA linked to your membrane?
DNA sequencing reagents
hi, thanks for reply!
my purpose is to extract total RNA with Tri..... separate on PA gel 8M urea, and transfert on hybon XL membrane (nylon)
I want to fix all RNAs (and siRNA in particular) in order to detect their presence.
I have never done a RNA transfers with 8M urea. This sounds similar to transferring DNA sequencing lanes to membranes for non-isotopic sequencing so it should work. Might be worth trying a small dot blot using your RNA in 8M urea first just to make sure the RNA is going to stick. This should also allow you to optimise the UV crosslinking time as well.
Longer automated DNA sequencing traces
You can bake your membranes (even nylon i,e. Hybond N+) at 80°C for 2h without any problem. In some cases, baking is preferable over UV fixation.
Put your membranes on wahtman piece, DNA face up, then let them to rest in the oven for 2h !!
The Zeta-Probe nylon membrane (Biorad) that I am using can be fixed via UV or 80 degree oven, though we've even skipped the fixation step and still had positive results.
thanks all.i'm gonna fix RNA by 5' UV at 254nm (but whithout knowing the energy of lamp) and bake it 2h at 80°. I'll see if it works and gonna tell you results.
Btw, i use Hybond XL membranes (nylon) so i assume it's ok as for Hybond N+.
Just remember that you can overfix and end up with very few accessible regions for the probe to bind. You might want to try doing either UV or baking.
I have always used UV for nylon and baking only for nitro-cellulose. I guess this is one of those myths that develop in labs. Good to find out that you can bake nylon too.
Automated DNA sequencing
in an old post, i told what i expect doing with membrane. Do you think bake is not suitable for siRNA detection?
I have no idea as I have never baked nylon before.
DNA sequencing analysis software