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- Label 4 tubes per sample, 2 as Cytoplasmic Extract (CE) and 2 as Nuclear Extract (NE)
- Chill tubes in freezer before use
- Trypsinize cells, spin down at 1.5k x g, aspirate trypsin
- Keep cells on ice for the full process
- Add 100 uL CE buffer w/ NP40
- Incubate 3 min on ice
- Spin 4 min at 1.5k rpm at 4 oC
- Remove CE to clean tube
- Gently wash nuclei in 100 uL CE buffer w/out NP40
- Spin as above
- Add 50 uL NE buffer
- Add 35 uL 5 M NaCl
- Add 50 uL NE buffer
- Incubate 10 min on ice, vortexing periodically to resuspend nuclei
- Sonicate NE
- Spin both CE and NE for 10 min at 21.1k g
- Transfer to fresh tubes
- Add glycerol to CE tubes to 20% (~22 uL)
- Freeze
