9 replies to this topic

[ram]

I have recently came across the protocol for RNA isolation by trizol method (Invitrogen: http://tools.invitro.....t 061207.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?
Thanks!

[phage434]

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.

[ram]

Thanks phage for the comments!

[gogreen]

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)

[ram]

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?

[phage434]

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.

[gogreen]

I normally don't see any liquid inside the tubes after a 3 min spin!

[ram]

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?

[gogreen]

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

[ram]

ok thanx for the info!