Hi,
I needed to prepare water saturated phenol for RNA extraction. I did as following: liquefy crystal phenol at 60oC, then add 100ml of liquefied phenol to beaker and followed by adding equal volume of DEPC-treated water, and mixed it by stir-bar. After waiting about 40min, I see three layers, the lowest is transparent, the more upper (middle) is milky, and the higest is transparent. I pipetted off two the uppers and repeated with second adding of equal volume of the lowest. After mixing and wating about 40 min, I could not see phenol phase. What did I do wrongly ? I think crystal phenol is in good condition.
One more, the water saturated phenol will used for RNA extraction. I did not treat container with DEPC , just autoclaved them. Is it safe for RNA working ?
P/S: I am a beginner, and poor for taking such experiment!
Please check the following link and PDF attachment.
http://openwetware.org/wiki/RNA_extraction_using_self-made_guanidinium-acid-phenol_reagents
Acid phenol- To solid phenol add RNase-free water until there is a layer of water on top of the phenol: Heat new bottle (500g) to 65 oC, crack lid. Add 100 ml of RNase-free water. Mix and let cool. Add about 100 mls more of water until a little water remains on top of phenol so that is it completely water saturated. Aliquot into 50 ml tubes and freeze at -20.
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