Question

How to extract only RNA from the solution in RNA prep? Which buffer is required to make it? Why DNA wouldn't be affected by that buffer?

Answer 1

Isopropanol is used for extraction of RNA. It helps in ectracting RNA over DNA since DNA is less soluble in isopropanol.  

Extraction of RNA:

• Take atleast 106 cells and wash with ice cold PBS after aspirating media.
• Remove PBS and add TRIzol.
• Transfer to eppendorf and incubate in RT for 5 minutes.
• Chloroform is added then and mixed well for 15 sec.
• Centrifuging at 10,000 rpm for 5 min leves three layers: Top aqueous, middle white contains DNA and bottom pink organic.
• Carefully remove top layer and transfer to seperate eppendorf.
• Add isopropanol and mix gently.
• Incubate for 5 minutes at RT.
• Centrifuge at 14,000 rpm for 20-30 minutes till the pellet barely visible at the base of tube.
• Aspirate IP and centrifuge with ethanol in DEPC treated H2O.
• Let the pellet air try and dry oout too much.
• Add either TE buffer or water to the RNA pellet.

RNA buffer contains TRIzol Reagent with 40%w/w Phenol, 1 M guanidine thiocyanate, 1 M Ammonium thiocyanate, 0.1 M sodium acetate buffer.