I have been working with RNA for sometime now using Trireagent and bead beating to lyse my cells and then when it comes to the step of phase separation, I keep seeing protocols mentioning 'add 0.2x chloroform into trireagent but no chloroform:isoamyl 24:1'. Is there any particular reason isoamyl alcohol is considered so bad for RNA? Is chloroform vs chloroform:isoamyl choice based on your starting material (dinoflagellates in my case) ?
The Tri-reagent is a mixture of phenol and guanidium thicyanate. After centrifugation step, the RNA is in the aquesous phase, DNA in the interphase, and the proteins in the lowest phenol phase. Aqueous RNA containing phase is then again washed with chloroform to remove any residual phenol. Phenol is more soluble in chloroform, compared to water, and both of these are more dense than water.
Isoamyl alcohol is sometimes included as an anti-foaming agent, but is generally thought to be an inert and optional addition.
Read this article. You'll understand better.
http://bitesizebio.com/3651/practical-application-of-phenolchloroform-extraction/
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