There seems to be salt in my RNA after extraction. I proceeded to perform a cleanup step and while the quality (260/280) improved, the 260/230 remains low. I do not have more sample to re-extract.
What are the concerns with this?
I'm sorry, but I need to disagree with Asif.
Trizol/phenol/guandinium peaks occur at *230* while nucleic acids are at *260*, which means a pure sample would have a higher ratio.
So, you definitely want a HIGHER 260/230 ratio 2.0-2.2.
http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf
May I ask what extraction, clean up, DNase, etc methods you use? I'm assuming a Trizol extraction?
Unfortunately, these contaminants do hinder cDNA synthesis and would probably compromise your downstream applications. I strongly suggest you start over, reprecipitate the RNA or go through another clean up process.
An additional chloroform extraction should remove any residual phenol and an ethanol wash should remove any chaotropic salts.
I add chloroform to my sample in Trizol, then add the mixture into phase lock tubes and spin. I then dump the aqueous phase into a new tube and add 70% ethanol before transferring to my RNeasy column. This is followed by manufacture's instructions (RW1 washes with DNase treatment in between, RPE wash, then 80% ethanol wash, and finally eluation).
May I ask where I should add the chloroforom and ethanol wash steps you're suggestion?
Thanks a lot!
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