"QUICK AND DIRTY" NUCLEAR EXRACTS
This is for up to 2 10cm plates of cells
1) Trypsinize as usual and spin down . Resuspend in 2ml of PBS and split into 2 microfuge tubes.
2) Spin 5 sec in microfuge (full speed) and resuspend in 1 ml of Buffer A.
3) Spin down again for 5 sec and resusupend in 1ml of Buffer A-set on ice for 10 min. to swell and lyse cells.
4) Vortex for 30 sec and check for lysis-if lysed spin for 5 sec and discard sups
5) Resuspend pellet in equal volume of Buffer C and set on ice for 15 min.
6) Spin 5 min at 40C in microfuge and take supernatant--aliquot and freeze at -800C
Buffer A (50 ml)
500ml 1M HEPES, pH 7.9
75ml 1M MgCl2
500ml 1M KCl
25ml 1M DTT
100ml 0.1M PMSF
49 ml dH2O
Buffer C (10 ml)
100ml 1M HEPES, pH 7.9
840ml 5M NaCl
15ml 1M MgCl2
4ml 0.5M EDTA
5ml 1M DTT
50ml 0.1M PMSF