Nuclear enrichment

互联网2013-11-13

1092

实验试剂

0.05% NP40 (or 0.05% Igepal or Tergitol)

pH 7.9

2. To prepare 250ml stock of Buffer A:

HEPES: 1M = 238.3g/litre, therefore 10mM = 0.59g/250ml

MgCl₂ : 1M = 203.3g/litre, therefore 1.5mM = 0.076g/250ml

KCl: 1M = 74.5g/litre, therefore 10mM = 0.187g/250ml

DTT: 1M = 154.2g/litre, therefore 0.5mM = 0.019g/250ml

4. To prepare 250ml stock of Buffer C:

HEPES: 1M = 238.3g/litre, therefore 5mM = 0.295g/250ml

MgCl₂ : 1M = 203.3g/litre, therefore 1.5mM = 0.076g/250ml

EDTA: 1M = 372.2g/litre, therefore 0.2mM = 0.0186g/250ml

DTT: 1M = 154.2g/litre, therefore 0.5mM = 0.019g/250ml

26% Glycerol (v/v) = 65ml

4.6M NaCl

87.66g/326ml H₂ 0

实验步骤

1. Requires large 12ml petri dishes. Prepare 1ml of Buffer A with added cocktail of protease inhibitors from a frozen stock and store on ice.

2. Add 500µl of Buffer A per large petri dish on ice and scrape thoroughly, leave on ice for 10 min (NP40 primarily lyses plasma membrane and leaves other membranes more intact than Triton X-100 does).

3. Centrifuge at 4^o C at 3000 rpm for 10 min.

4. Remove supernatant and keep (this will contain everything except large plasma membrane pieces, DNA, nucleoli), extract out 10µl for Bradford assay.

5. On ice, resuspend pellet in 374µl of Buffer C and add 26µl of 4.6M NaCl to give 300mM NaCl (high salt helps lyse membranes and forces DNA into solution).

6. Homogenise with 20 full strokes in Dounce or glass homogeniser on ice.

7. Leave on ice for 30 min

8. Centrifuge at 24,000g for 20 min at 4^o C

9. Aliquot supernatant, remove 10µl for Bradford assay and store at –70^o C.

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