I'm using the mirvana mirna isolation kit with phenol chloroform. I'm having a difficult time discerning the phase separation upon addition of acid phenol chloroform and centrifugation. I've attached a picture of a sample after centrifugation. Is the aqueous phase the entirety of the upper part of the solution, while the organic layer is the solution that settled at the bottom? I don't seem to see any interphase layer that's why I'm in doubt.
Thanks for anyone who could help me out
Did you use RNA later or a high salt solution to prepare the cells in? High salt can disrupt phase separation.
Can you provide a detailed procedure. Which kind of cells, what did you do to the cells prior to adding lysis buffer? what volumes of the reagents you used up to the acid phenol step?
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