ARGH! Northern Problems (again!)
aaron at ccat.sas.upenn.edu
Thu Jan 18 15:05:34 EST 1996
Several possible reasons for this here. First, spec. readings aren't the
most reliable things in the world all the time. If you want VERY even loading,
it's probably a good idea to run a gel (if possible) with 2 ug of RNA, and then
readjust your concentrations based off of relative intensity.
My preferred method for dissolving dried RNA (and I DRY it) it so to heat it
to 55C for 10 minutes, vortex, spin down, heat for 10 minutes, vortex, spin
down and freeze. Works great, no degradation. I also redissolve my RNA in
TE (pH 6.8) / 0.1% SDS.
Skip the second drying step. Try and dissolve your RNA so that it's in a
workable concentration. If you can't do that, I'd suggest just using the
Speedvac to reduce the volume of the sample.
Patrick HJ Falckh (p.falckh at rmit.edu.au) wrote:
: Robert Fisher <fisher at radonc.unc.edu> wrote:
: >After months of successful northern blots, i have run into a problem. When
: >i run my total RNA out on a 1.2% agarose/formaldehyde gel, the 18S and 28S
: >vary in intensity from one sample to another, although according to the spect.
: >a260 readings i am loading the same amount of RNA. There doesn't appear to be
: >much degredation. The RNA is isolated by the GITC/phenol method.
: >This may seem silly, but could this be due to a solubility problem? I don't
: >overdry my RNA pellets from the EtOH ppt, and I resuspend them in DEPC h20. I
: >usually let them dissolve and leave them at 4 degrees c for an hour or so
: >before i freeze them down. I measure out the proper amount of RNA solution
: >(according to our spect.) to give me 10ug, then dry it down in the speedvac.
: >Again, i am careful not to overdry. i resuspend in denaturing loading buffer
: >and heat at 95 degrees for 5 minutes to denature prior to loading my gel.
: >can anyone see anything obviously wrong?
: >-rob fisher
: Why dry it down a second time. Provided that your initial solubilisation is not too dilute why not add the denaturing loading bu=
: ffer to a small aliquot of your original solublised RNA. A method out of FOCUS (10 I think ?!) allowed the use of 4.5 ul of sample =
: (either the required amount of RNA in that volume or add SDW to make the volume 4.5 ul) to which was added 15.5 ul of sample buffer =
: which was made up as :
: 1.0 ml deionised formamide
: 350.0 ul formaldehyde
: 200.0 ul 10x MOPS
: - then aliquot into 300 ul units to store at -20C.
: If you add 1 ul of 1.0 mg/ml ethidium bromide and THEN heat denature, then add 4.0 ul of loading buffer (6x stock). The sample load=
: ed can be visualised on the gel after running without further staining or other treatment and the RNA is approx. 10x as bright compa=
: red to adding the EtBr after denaturing.
: This saves heaps of time and there is little chance of other difficulties affecting the result
: Hope this ios helpful
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