Messy RNA extraction - Trizol protocol, odd pellet (Apr/16/2008 )

Hello,

I'm using trizol to extract RNA from catepilars, The aqueous phase after chloroform addition has a yellowish/orange colour. I read in the troubleshooting it might be due to fat and miselles trapping Trizol. I'm currently after the LiCl step with a huge yellow pellet that should be small and white. What can I do to my sample now? Shall I start over?

i used to get a yellow pellet like you did. but the nanodrop and bioanalyzer result is still very good.
by the way, did you try to look at your nanodrop or bioanalyzer result?

Not yet, I tried to keep working on it but after resuspending my pellet in 20 microliters of DEPC treated water and heating to 70°C to get rid of the ethanol it became jelly-like and I cannot pipette it anymore, I was told I should leave it overnight with a bit of water.

I'm growing confused now.

BTW, Sanjiun81, from what kind of tissue did you get this yellow pellet from?

Have you tried reducing the amount of tissue you use in the RNA extraction? For difficult samples it helps working with decreased amounts of starting material to get a better quality result.

We have had a similar problem in the lab recently. It seems / we believe the isopropanol was the problem -it was in plastic bottle and I don`t remember the company`s name. Now, we use isoprop from Merck (dark 2.5lt glass bottle) and we don`t have that problem anymore.

i use same brand of isopropanol but i only got yellow pellet once in a while.
I use cell line. normally i'll put 6ml trizol/tri-reagent in a 75T flask.