Question about TRIzol Extraction - (Feb/25/2008 )

Hi everyone,

I would like to ask questions about the TRIzol Extraction.

Basically, I follow the protocol stated by invitrogen.

However, after I use the 75% Ethanol to wash white pellet/ transparent gel like pellet and centrifuge down, no pellet is observed. I would like to ask if it is normal, if yes, how I can remove the supernatant? As I scared I will decard the RNA, how I can remove the supernatant without affect the RNA?

Thanks

if you spin long and high speed enough... it is very hard for your pellet (if any to detach)... after spinning down, i'll gently invert the tube and pour the ethanol out on tissue (better to absorb the excess EtOH). I shouldn't detach if you do it gently.

DO NOT remove supernatan using pipette/tips you might accidentally touch the pellet. after pouring out the supernatan, leave it to air dry at RT or use a little heat on heating block. EtOH shall evaporate fast.

THANKS~~

And I would also like to ask if I want to check the integrity of the RNA.
Does it ok, by 1% agarose gel electrophoresis with EtBr staining?
I found that someone heat inactivated the RNA sample before running the gel, what is it for?
Also, what is the minimium amount of RNA (ng) in order to be detected by gel electrophoresis?
One of my sample have only 62ng/ul and 20ul...
so I want to know if 5ul is enough for the detection.

Thanks a lot~

I don't know what your down stream applications are but I usually add RNA carrier to my extractions. something like 25ug of yeast tRNA. You will definitely see a pellet then.

Agarose gels are only recommended for very large RNAs. The pore size is to large to resolve anything under 500BP very well.