Is it only water and nucleic acid in the top phase? - (Mar/15/2012 )
After treating cDNA with RNase H I did a phenol/chloroform extraction to purify my cDNA, however I forgot to add isopropanol to pellet down the cDNA, and I used the top phase directly in PCR and surprisingly I got PCR product.
what is the component of top phase during phenol/chol extraction? is it just water and nucleic acid??
I think whatever is soluble in water. But there probably weren't much of these things in cDNA+RNaseH reaction.
my RNase H reaction is only 20 ul and I normally add some additional 20-30 ul water just before adding 100 ul phenol/Cholo. The more water I add, the bigger the top phase.
I'm not sure if this applies to Trizol extraction too. I think the top phase that I get during RNA extraction with Trizol is bigger than the volume of my sample in water.
the top phase is the aqueous phase and consists of water soluble components of your solution (minus the proteins which are precipitated by the phenol). it will include buffer from the phenol.