Methods in molecular genetics

• Draw 5 ml of blood and mix with EDTA anticoagulant

• Keep cool until prep is performed (do not freeze)

• Transfer 2 ml blood into a 15 ml Falcon tub, add 10 ml of Red Cell Lysis Buffer (RCLB)*¹ and mix completely by inversion

• Spin 10 min @ 1,200 x rpm (clinical centrifuge)

• Discard supernatant and resuspend cell pellet in 10 ml RCLB*¹

• Spin 10 min @ 1,200 x rpm

• Resuspend white cell pellet in 0.4 ml of White Cell Lysis Buffer (WCLB)*² (pipette up and down carefully!)

• Final extraction, add 150 µl of saturated NaCl (~ 6M NaCl) to 400 µl of white cell lysate

• Vortex and invert to mix; place on ice for 10 min

• Centrifuge 5 min @ 12,000 rpm (if there is no clear pellet - add some more NaCl and repeat this step)

• Add the supernatant (550 µl) to a new tube, add 1ml 100% ethanol. Mix by inversion - DNA will precipitate

• Transfer DNA with sterile pipette-tip into new tube

• Wash once with 500 µl 70% ethanol

• Spin 20 sec @ 12,000 rpm; discard supernatant

• Dry DNA pellet for 5 min at 37°C (do not dry too long or it will be difficult to redissolve the DNA !!!)

• Resuspend in 200 µl of TE.  Leave ON at 4°C to resuspend completely

• *¹ 1 x RCLB: 1 mM NH₄CO₃, 115 mM NH₄Cl (autoclave 10 x buffer)

• *² 1 x WCLB: 100 mM Tris-Cl (pH 7.6), 40 mM EDTA (pH 8.0), 50 mM NaCl, 0.2% SDS, 0.05% NaN₃