| DNA ISOLATION FROM PRIMARY TUMORS&nb | | 点击： 作者： 来源： 时间： 2006-11-06 本站论坛 |
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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
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