Doubt: Trizol protocol - about certain steps (Dec/01/2009 )
I have recently came across the protocol for RNA isolation by trizol method (Invitrogen: http://tools.invitrogen.com/content/sfs/ma...t%20061207.pdf). I have tow doubts about the protocol.
1) It has been told to not to take tissue more then 10% of the volume of Trizol reagent used. Why? What would happen if more tissue is used ?
2) After isopropanol precipitation, to harvest the RNA pellet it is stated to not to exceed 12,000 g speed. Why?
3) "Do not dry the RNA by centrifugation under vaccum" Why?
1. Inadequate reagent to fully process the cells in your tissue sample. This could lead to sample contamination with unprocessed tissue components.
2. Too compact a pellet for resuspension? Break the tubes? Precipitate other stuff like proteins?
3. Totally dry DNA or RNA is very difficult to resuspend, taking many days.
Thanks phage for the comments!
1. Maybe they'd have tried various amounts of starting materials and amounts of Trizol and found that this ratio works best. But lately I tried using the same amount of trizol for a 50 ml and a 100 ml yeast culture and ended up with linear yields..so I believe its quite flexible
2. I do the precipitation and washes at max speed (14k g)...never had a problem, so I wonder why they say it!! maybe as phage said, to avaoid compaction of the pellet!
3. I'd read it as "Dont vacuum dry the RNA for too long" (I do a 3 minutes vacuum spin after the ethanol wash and resuspend the pellet asap after making sure there is no more ethanol leftover)
How do you make sure there is no more ethanol leftover? By smelling? Because there would be some water leftover. Everything wont dry in just 3 min, right?
Ethanol is quite volatile. A few minutes with an open tube on the bench is usually adequate to evaporate most of it. A vacuum will make this very quick indeed. A small amount of ethanol will also probably not provide problems in most applications.
I normally don't see any liquid inside the tubes after a 3 min spin!
Do u use conical bottom tubes (1.5ml) or parallel wall (2ml)? Do you tap the tubes after inverting to remove as much liquid as possible after pelleting?
I use 1.5 ml conical tubes and I don't decant the solution during the isopropanol precipitation and ethanol wash, I pipette it out to make sure that I remove most of the liquid in first place
ok thanx for the info!