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"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 4
0C in microfuge and take supernatant--aliquot and freeze at -800C
Buffer A (50 ml)
500
ml 1M HEPES, pH 7.975
ml 1M MgCl2500
ml 1M KCl25
ml 1M DTT100
ml 0.1M PMSF49 ml dH2O
Buffer C (10 ml)
100
ml 1M HEPES, pH 7.92.5ml glycerol
840
ml 5M NaCl15
ml 1M MgCl24
ml 0.5M EDTA5
ml 1M DTT50
ml 0.1M PMSF6.4ml dH2O