northern of 450 and 500 bases - agarose or polyacrylamide? (May/10/2006 )
i have to set up a northern blot for resolving sizes around 500bases.
I've done agarose formaldehyde gel, but it seems i have to redo the gel (with the SAME samples)
and therefore, as it's more convenient to proceed with an acrylamide gel, i want to know what is your opinion (especially regarding the percentage of the gel. I would use a 8%acryl...)
I don't want to compare between an other experiment. in fact it's because with an agarose gel, i can only use a capillary transfert, and it's a crazy thing to set up... (in fact twice, the thing fall off...). With acrylamide, i can do a electrotransfert, which is quicker (2h at most). Sosaving time and be more sure of the transfert....)
maybe a 6% gel. that's what we used for sequencing and 500 bases were still pretty high on the gel.
ah... so i assume that's not a good choice to test on PAGE gels?...
maybe not the best choice. you could use a lower % acrylamide (4 or 5%), run longer, run a sequencing gel. keep in mind that these are tbe buffered gels (and get hot). otherwise page is normally used to purify smaller (than 500 bases) oligos.
thanks a lot mister mfdenko.
you know, you could try it. i actually kind of like the idea of a 6% gel (8% may be to restrictive for a small gel). your two rnas should be about 148 and 165 kDa. they should separate reasonably.
nothing ventured, nothing gained.