DNA ISOLATION FROM PRIMARY TUMORS VIA CRYOSECTIONS

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) :