Talk:RNA extraction Trizol

This protocol has been adapted for 500 ml Ehux culture (10 *6 cells/ ml) Decalcification step (cf RNA extraction GITC protocol) Harvest the cells by centrifugation 10 min at 10 000g Remove the supernatant Resuspend the pellet in 1ml Trizol (the sample can be kept frozen in liquid nitrogen or -80°C at this step) Resuspend the pellet using a 21 G seringe Incubate 5 min at room temperature Centrifuge 10 min, 12000g at 4°C to pellet the membrane debris and polysaccharids Save the supernatant in a clean tube Add 200 µl chloroform per ml of initial volume of Trizol Shake vigourously 15s Incubate 2 min at room temperature Centrifuge 15 min, 12000g at 4°C Save the aqueous phase containing the RNA Add 200 µl of chloroform Centrifuge 10 min, 12000g at 4°C Save the aqueous phase in a clean tube Add 500 µl isopropanol per ml of initial volume of Trizol Invert the tube to mix Incubate 15 min at room temperature Centrifuge 15 min at 12000g Remove the supernatant Wash the pellet in ethanol 75 % (1 ml per ml of initial volume of Trizol) Mix by inversion Centrifuge 5 min, 12000 g at 4°C Remove the supernatant Resuspend the pellet in 40 µl treated water Shake the tube about an hour at 4°C