DNA ISOLATION FROM PRIMARY TUMORS VIA CRYOSECTIONS - make a 5μm section to do an evaluation of the % tumour cells - make 50X50μm sections for the DNA isolation - put sections in a falcon tube containing 10 ml of 1XSE - add 100μg/ml prot. K (endconcentration) (1 ml of 1mg/ml in 10 ml of SE buffer) + 1% SDS (endconcentration) (stock is 25% SDS) - put the tube o/n at 55°C - add 1/4 of the volume (2.5 ml) of 6M NaCl(saturated solution, precipitation at the bottom) - forming of precipitate in the tube, mix gently by inverting the tube a few times. - add 1 vol chloroform (stabilized with ethanol) (12.5 ml) and invert the tube a few times - put tube for 1 hr on rotator at room temperature - centrifuge 10 min 2000 rpm - forming of 2 phases, phase at the bottom is chloroform, middle phase contains proteins, SDS,...., top phase contains DNA - transfer of the top phase to another tube by means of a pipet tip (cut off) - add 1 vol isopropanol - invert the tube a few times, thread of DNA will form, if not centrifuge for 30 min 4000 rpm at 4°C - remove thread of DNA with a closed hooked pasteur pipet and rinse gently in 70% EtOH - DNA is dried and dissolved in appropriate volume of TE buffer - dissolve the DNA o/n at 4°C on rotor SE buffer (10X) :

750 mM NaCl pH=8 250 mM EDTA

?