Extraction d'ADN

Get a fresh culture of E. huxleyi (e.g 500 ml) Centrifuge 10 min at 20000 g Add 50 to 100 µl og GITC Buffer* Mix it gently using fingertip knock Keep it frozen (-20°C) if you want to stock the sample or proceed the following instructions Add a volume of phenol pH 8 Incubate for 5 min at RT Save the aqueous phase in a clean tube Add 1 volume of phenol-chloroform-alcool isoamylic (25:24:1), invert the tube three times Incubate 5 min at RT Centrifuge 10 min at 8000g Save the aequous phase in a clean tube Add 1 volume of chloroform Incubate 5 min at RT Centrifuge 10 min at 8000 g Save the aqueous phase in a clean tube Add 1/10 volume sodium acetate 3M pH 5.2 and 2 volume of 100% ethanol Keep the tube from 30 min to 1 hour at -80°C or ON at -20 °C Centrifuge 20 min at 9000 g Remove the supernatant Wash the pellet in 500 µl 70 % ethanol Centrifuge 10 min at 7500 g Remove the supernatant Resuspend the pellet in 50-100 µl sterile water Keep it on ice for 1 hour at 4 °C

GITC buffer: 100 g guanidine thiocyanate in 100 ml distilled water 10.6 ml 1M Tris-HCl (pH 7.6) Stir ON at RT (it takes usually several hours, don't forget to put a cover when stirring the solution) Add 21.2 ml 5% sarkosyl and 2.1 ml beta-mercaptoethanol Adjust the volume to 212 ml in distilled water Store at 4°C in a brown bottle or wrap in aluminium foil