5 replies to this topic

[UBClabbie]

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!

[Curtis]

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.

[UBClabbie]

Curtis, on 05 November 2010 - 09:27 PM, said:

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.

Hmm everytime I do the extraction I only take about 1/3 of the upper phase to avoid contamination from the interphase and lower phase.
However, I'm still getting a low ratio.

[UBClabbie]

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

[RecklessTech]

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

[UBClabbie]

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

RecklessTech, on 17 November 2010 - 01:27 PM, said:

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