RNA pellet white and low ratio (low purity) - how to improve RNA purity (Nov/05/2010 )

Hi,

I've been performing RNA extraction using Trizol.
I follow the manufacturer's protocol of adding 1ml Trizol per 10cm2 dish, add 200ul chloroform, isopropanol and wash with ethanol (and of course all centrifugations required in between)

However, I keep getting a whitish pellet, which my supervisor told me means that there is a high unwanted salt content.
I also keep getting A260/A280 ~ 1.48. The best I ever obtained was 1.6 =/

I'm wondering how I can reduce the salt concentration in my RNA pellet and improve the RNA purity.
Total RNA yield is never a problem.

Questions:
Will adding more Trizol help?
Will adding more isopropanol help?
One forum suggested washing with EtOH more than once.


Thanks!

After addition of chloroform you get three phases. just save the upper phase and make sure your pipet tip doesn't touch the interphase. You can alternatively just save half of the volume of the upper phase and check the purity.

Curtis on Sat Nov 6 05:27:45 2010 said:

Update:

I decided to spin down my sample after trizol addition and prior to adding chloroform, as suggested by the Trizol protocol. I also performed the EtOH step twice (as I've seen this suggested elsewhere on the web) and got a much better ratio of 1.68

My pellet looked less white and more gel like when I added these steps

What is your percentage of Ethanol? Higher concentrations don't allow for salt-h2o exchange in the wash step.

the Trizol protocol that was supplied says to use 75% EtOH, but i've been switching been that and 70%

RecklessTech on Wed Nov 17 21:27:53 2010 said: