I have been having problems isolating mRNA lately. Previously, I had no problems at all. When I isolate my total RNA from S. cerevisiae the OD260/280 ratio is always good (between 1.9-2.1). I use oligo (d)T cellulose to extract poly-A RNA from the total RNA. When I measure the OD ratio for the mRNA eluted from the columns the ratio is always 1.2 or lower. This has been the case since we purchased a new stock of oligo (d)T. This has only happened one time before. The first time my advisor showed me how to isolate Total/poly-A RNA. When I redid the experiment using a new stock of Oligo (d)T it worked fine. We have since ran out of that stock and I am working with a new one. I don't know if the oligo (d)T is the problem or not. I think I am wondering what could be happening between isolating the total RNA and when I isolate the poly-A RNA that could cause the mRNA to be degraded. I do use DEPC treated tips, pipets, reagents and wear gloves. My water is always DEPC treated or nuclease free water (purchased) but I can't say how careful other students are with the nuclease free water. The only other thing I can think of is that my work area might be contaminated. Previously I used an area in the lab that no one ever walked by. Since it is now another students personal desk, I can't use it. How can I create an RNase free environment (or should I just put on a mask and a biohazard suit)?
I am assuming the RNA is degraded because when I do RT-PCR or 5' RLM-RACE I get a huge bright smear instead of a discreet band(s).
she also says it isn't the water,
thank you for any help provided,
Edited to add :
she also just said this to me, "if they ask. it isn't the reagents, tips, etc. and I do wear gloves. I have tried RNasins"
Edited by Hellfire, 16 August 2004 - 07:59 PM.