A few questions on RNA extraction and stability - (Oct/22/2005 )

I just use Tris (10mM, pH 8.0) or TE (ala Maniatis, pH 7.6)

I don't know if the salt will affect downstream stuff? It shouldn't hurt your spec though


My question regarding the spec was not whether the readings were close to zero, but whether you feel you can trust your spec. Those sensitive little buggers can make or break your experiment...and are your cuvettes good?

Hmm... Tris, TE or DEPC water seem like good choices. I will probably place an order for some TE, and make sure that it is specifically in the 7.6-8 range.

As for the spec, it is a fairly new one, and I am using a standard cuvette. Acutally, now that I think about it, I filled it up near the clear portion where the light shines through i.e. it was not completely filled. It is possible that the meniscus could have been in the upper edge. This seems like the most reasonable explanation for my crazy results. Possibly one of the readings was closer to the rim than the other. Could this create the variability that I am seeing?

I did this to minimize the dilution, since before I had barely gotten any RNA. From this point on, I will make sure to fill the cuvette above the clear window.

Hi, i am also newbie for RNA extraction. I had extracted RNA and run on 2% TBE gel. But it has a very serious smear along the RNA sample lane. I attacted the photo... Please give some guidance for how should i ratify the smear and is it possible to perform q-RT-PCR.