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The Laboratory of Donald Rio at the University of California, Berkeley
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| Expect about 3.4 ml of extract for every 4 liter of cells (depending on initial cell concentration). |
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1. Perform preparations on 4 to 32 liter of cells at about 3.5 X 106 cells/ml (see Hint #1).
2. Harvest the cells in two 1 liter bottles with repeated centrifugation in Sorvall RC3 rotor at 4°C at 2000 rpm for 10 min.
3. Resuspend the cells with a wide-bore pipette in a total of 50 ml cold PBS with 1 g/liter MgCl2 (see Hint #2).
4. Transfer the cells to 50 ml conical tube pre-cooled on ice and centrifuge in a Beckman Accuspin at 4°C at 2300 rpm for 10 min to pellet cells.
5. Estimate the packed volume of cells. Resuspend pellets in 5 packed-cell volumes of DR Buffer A and incubate the tubes 10 min on ice.
6. Pellet cells as in a Beckman Accuspin at 4°C at 2300 rpm for 10 min.
7. Resuspend cells in 2 packed-cell volumes of DR Buffer A.
8. Lyse cells with 10 to 15 strokes in a dounce homogenizer with B (tight-fitting) pestle (see Hint #3).
9. Transfer lysed cells to round-bottom polycarbonate tubes.
10. Centrifuge at 4°C, 2300 rpm in a Beckman Accuspin to gently pellet the nuclei.
11. Pour off most of the supernatant (see Hint #4).
12. For S100, Add 0.11 volumes of DR Buffer B to supernatant, Centrifuge at 35,500 rpm at 4°C for 1 hr in a 45Ti rotor (100,000 X g).
13. Remove lipid layer at top, if possible, and dialyze against 20 volumes of DR Buffer D with 3 changes of Buffer.
14. Pack nuclei in a Sorvall SS34 rotor for 20 min at 4°C at 14,500 rpm (25,000 X g).
15. Resuspend nuclei in 2.5 ml of DR Buffer C.
16. Place the nuclei in a dounce homogenizer and stroke 5 to 10 times with pestle A (loose-fitting) until the nuclei are resuspended.
17. Transfer nuclei with plastic pipette to round-bottom polycarbonate. Rotate at 4°C for 45 min.
18. Centrifuge the nuclei in an SS34 rotor at 15,000 rpm (27,000 X g) for 30 min at 4°C.
19. Dialyze nuclear extract for 5 hr against DR Buffer D (at least 50 volumes) with changes to fresh DR Buffer D after 1 hr and another change after 2 hr.
20. Centrifuge the nuclei in an SS34 rotor at 12,000 rpm (17,000 X g) for 10 min at 4°C to remove precipitates.
21. Freeze 50 to 100 μl aliquots in 0.5 ml microcentrifuge tubes in Liquid Nitrogen.
22. Test conductivity and protein concentration (see Hint #5).
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| DR Buffer D |
| 0.5 mM PMSF
0.2 mM EDTA
20% Glycerol
pH HEPES with KOH, add DTT and PMSF to solution just before use
0.1 M KCl
20 mM HEPES, pH 7.6
0.5 mM DTT
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| DR Buffer C |
| 0.5 mM PMSF
0.2 mM EDTA
pH HEPES with KOH, add DTT and PMSF to solution just before use
20 mM HEPES, pH 7.6
0.5 mM DTT
1.5 mM MgCl2
25% Glycerol
0.42 M NaCl
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| DR Buffer B |
| 0.3 M HEPES, pH 7.6
pH HEPES with KOH
0.03 M MgCl2
1.4 M KCl
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| DR Buffer A |
| 10 mM HEPES, pH 7.6
10 mM KCl
0.5 mM DTT
1.5 mM MgCl2
pH HEPES with KOH, add DTT to solution just before use
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| PBS with MgCl2 |
| Add 1 g/ml MgCl2
pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
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HEPES
PMSF
Potassium Phosphate, Monobasic
Potassium Hydroxide
EDTA
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
DTT
Magnesium Chloride
Glycerol
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1. Preparation described is for 4 liters. The state of the cells is crucial. It is important to split the cells very carefully when they are growing in T-flasks; specifically, they should be maintained at a fairly high density. Let them grow to confluency and then split them 1:5. The cells should reach confluency again in three days.
2. Keep 1 liter bottles on ice and use a cold pipette. All equipment referred to after this step is used after pre-cooling at 4°C overnight. All steps should be performed on ice at 4°C (in a cold room).
3. Check lysis in microscope, expect about 90% lysis.
4. Be careful not to pour off the nuclei which are only loosely packed at this point (This step removes lysozymes).
5. Expect about 0.13 M KCl, 10 to 12 mg/ml.
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1. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983; 11:1475-89.
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