RNA pellet white and low ratio (low purity)
Posted 05 November 2010 - 02:03 PM
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.
Will adding more Trizol help?
Will adding more isopropanol help?
One forum suggested washing with EtOH more than once.
Posted 05 November 2010 - 09:27 PM
Posted 08 November 2010 - 10:33 AM
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.
Posted 09 November 2010 - 03:44 PM
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
Posted 17 November 2010 - 01:27 PM
Posted 24 November 2010 - 02:50 PM