RNA migration in agarose formaldehyde gel - (Oct/17/2006 )

I ran my 1 ug RNA samples (including millennium markers and control RNA) on a 1% agarose formaldehyde gel at 60V. I also had the Ambion formaldehyde load dye in my samples.
For all the samples, the RNA did not migrate, while the bromophenol dye migrated.

I included 1 uL 10mg/mL EtBr in the samples instead of including EtBr in the agarose. Is it possible that the EtBr impeded migration of the RNA?

James

Hey,

Nope. EtBr won't impede your RNA significantly.
Did your ladder migrate?
If not, your electrophoresis tank is not carrying current.
If your ladder ran, your RNA may have alot of protein contamination,
or alot of genomic DNA. These will impede your migration.
Or possibly, you do not have RNA, but have mistakingly isolted genomic DNA???

EtBr shouldnt prevent ur samples from migrating.

As mikew suggested, u have a problem somewhere else.

I always add EtBr to my RNA samples, it should not affect migration. One obvious difference I noticed between your gel and how I run mine is that I run mine at 240V (yes, that's right, 240V). It'll be done within twenty minutes or so, but for some reason it works really well. Anyway, good luck

Thanks for answering.
Nope the ladder did not migrate either.
I do not think it is a problem with sample contamination because both control RNA (Ambion) and ladder (Millennium Markers) did not migrate either. I have never used this electrophoresis tank before; however I did see bubbles... but maybe the voltage was too low (60V for a ~ 11 cm gel).

So, the bromophenol dye (in the formaldehyde load dye) migrates even in the absence of current?

hi greenpixie

how much EtBr do you add to your samples? I am thinking of just adding 1uL of 10mg/mL to the 100mL gel.
I will rerun my sample today at 80V and see what happens!

that's the same amount i use--just one microliter for each sample
good luck