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Hey guys, I suspect that I need to change the protocol for the RNA extraction.
I am working with small amount of tissue - may be 3-6 mg.
And the protocol that I was advised to use suggest me to use 1 ml of Trizol.
I think it is wayyy too much, because I end up with 260/230 about 0.3-0.9 which implies phenol contamination
Can you please advise me what would be the appropriate amount of Trizol in my case?
Re: Trizol question
See the following protocol for Trizol RNA extraction.
[Only registered users see links. ]
BTW the 1 mL Trizol does not affect the final concentration.
You precipitate the RNA, then the resuspension buffer volume is going to determine final concentration.
If you truly have phenol contamination (after isopropanol precipitation and EtOH washes) you need to work on your lab techniques especially with the micropipette.
Re: Trizol question
Thank you for the reply and the protocol!
I was not complaining about my final RNA concentration - that one is fine.
It is the 260/230 ratio that bothers me, it should be 1.8 and higher, and mine is wayyy lower.
The reason why I suspect that tissue amount is the issue for 1 ml of Trizol, is because when I am handing larger amounts, e.g. 50-100 ug, no probem, 260/230=2.2
The same problem is shared by 3 other people in the lab who work with small tissues, so it can't be all bad lab skills...
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