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.
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.
Hi David, I am starting with Trizol extraction, but taking the aqueous phase and continuing with RNeasy extraction. I am doing in-column DNase treatment.
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?