Extract Preparation Protocol
Cytoplasmic Extract Protocol Sheet
| Date:_____________________________ Cell type:__________________________ Media:____________________________ Cells/ml:___________________________ Culture volume (ml):_________________ |
Treatment before extraction: (eg TPA -4hr) |
Take 10ml sample for RNA analysis. Spin down cells at 300g in Sorvall Resuspend pellet in ice cold 1 x PBS (15-50ml) Transfer to conical tube and spin 300g in benchtop centrifuge, 5 min Resuspend pellet in ice cold 1 x PBS (15-50ml) Spin 300g in benchtop centrifuge, 5 min Note packed cell volume: Packed Cell Volume:_______ µl Resuspend in 5PCV buffer A + DTT, PMSF Buffer A [KCl]:___________ mM Buffer A swelling (5 x PCV)________ ml Swell on ice 10 min Spin 300g in benchtop centrifuge, 5 min Carefully remove supernatant. Add 2PCV Buffer A Buffer A lysis (2 x PCV)__________ ml Lyse by douncing 10 times on ice with loose pestle. Spin 2000rpm in benchtop centrifuge, 5 min Take the supernatant to a fresh tube. Estimate extract volume. Extract volume:_________ µl Add buffer B (0.11 vol for 10mM buffer A, 0.09 vol for 50mM buffer A) Buffer B volume:_________ µl Spin 100000g for 1 hour. Remove supernatant to fresh tube. Estimate extract volume. Extract volume:__________ µl Add 0.25 volumes 80% glycerol. 80% glycerol volume:_________ µl Final extract volume:__________ µl Final extract concentration:_________ mg/ml Storage location:___________
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