Cepko/Tabin Lab ,Harvard University
http://axon.med.harvard.edu/~cepko/protocol/mike/N1.html
This is the standard protocol with only minor modifications. The volumes indicated are for 6 liters of cells at 1-2 x106 cells per ml.
Solutions
Solution A (Equilibration Buffer: 100 ml)
10 mM HEPES 7.9 (pH with KOH) 1 ml 1M HEPES pH 7.9
1.5 mM MgCl2 150 ml 1M MgCl2
10 mM KCl 1 ml 1M KCl
up to 100 ml with Q
for every 100 ml add:
100
ml .5 M DTT
500
ml PMSF (Sigma #P7626)
100
ml pepstatin (2.0 mg/ml)
100
ml leupeptin (1.0 mg/ml)
100
ml aprotinin (5.0 mg/ml)
(Protease Inhibitor Cocktail Sigma #P8340)
1.0 ml 0.1 M EGTA
1.0 ml 10% NP40
Solution B
(Low Salt Buffer: 100 ml)
20 mM HEPES 7.9 2.0 ml 1M HEPES 7.9
1.5 mM MgCl2 150 ml 1M MgCl2
20 mM KCl 2.0 ml 1M KCl
0.2 mM EDTA 40
ml .5 M EDTA
up to 100 ml with Q
for every 100 ml add:
100
ml .5 M DTT
500
ml PMSF
Protease inhibitors (see Solution A)
1.0 ml 0.1 M EGTA
1.0 ml 10% NP40
Solution C
(High Salt Buffer: 10 ml)
20 mM HEPES 7.9 200
ml 1M HEPES 7.9
1.5 mM MgCl
2 15 ml 1M MgCl2
1.2 M KCl 3.0 ml 4M KCl
0.2 mM EDTA 4
ml .5 M EDTA
up to 10 ml with Q
for every 10 ml add:
10
ml .5 M DTT
50
ml PMSF
Protease Inhibitors (see Solution A)
100
ml 0.1 M EGTA
100
ml 10% NP40
Solution D
(Dialysis Buffer: 2 liter)
20 mM HEPES pH 7.9 40 ml 1M HEPES 7.9
100 mM KCl 14.9 g KCl
0.2 mM EDTA 800
ml 0.5 M EDTA
20% glycerol 400 ml glycerol
up to 2 liter with Q
for every 2 liters add:
4.0 ml 100 mM PMSF
2.0 ml 0.5 M DTT
Procedure
GENERAL NOTES: I try to gently dislodge the pellet after every step to avoid pipeting up and down to resuspend the cells or nuclei. If a given spin is not sufficient to pellet the cells or nuclei I have found a quick spin at higher rpms is useful (such as 5’ at 1.5-2K).
• Cool the SA600 rotor with the small adaptors to 4° and cool the 50 ml tube adaptor for the J6B.
• Harvest cells in 1 liter bottles by spinning in J6B at 1.2K for 30' @ 4
o. Pour off most of the medium and as the cells come loose, stop. Pool the remaining medium and cells into 2-4 250 ml conical bottles and spin at 1.2K for 10 minutes. Gently resuspend in 4x 50 ml HBSS, transfer to a 50 ml tube, and spin in the J6B for 5 minutes at 1.2K. Aspirate the HBSS and gently flick the tube with your finger to dislodge the pellet.
• Resuspend the pellet up to 2x the pellet volume of Solution A and pool into 2x 50 ml tubes. (I generally get 15-25 ml cells total and resuspend up to 30 to 50 ml).
• Spin in the J6B for 5' at 1.2K and gently flick the tube to dislodge the pellet, then resuspend in the remaining Solution A (there should be about 70-80 ml left). Equilibrate on ice for 20'. The Solution A plus the cells should be about 100 ml.
• Take a look at the cells with Trypan Blue to see if they have already lysed. If the cells still appear to be intact, dounce approx. 30 ml 10 times and recheck with the Trypan Blue. Repeat this until >90% of the nuclei take up the dye.
• Spin in the J6B for 5' at 2K and aspirate the supernatant. Flick the tube with your finger to gently dislodge the pellet (it may be a little sticky at this point).
• Gently resuspend the pellet up to 40 ml final volume with Solution B and spin in the J6B for 5' at 2K. You may lose some nuclei here, but it is better than packing them too tightly.
• Remove the supernatant and flick the tube with you finger to gently dislodge the pellet.
• Resuspend the nuclei in 0.5 volumes of Solution B and slowly add 0.5 volumes of Solution C (Add it dropwise with a pasteur pipet while vortexing on 3 or 4).
• Mix on the tiltboard for 30' @ 4
o.
• Transfer the nuclei into a 15 ml glass Corex tube and spin in the SA600 for 30' at 10K.
• Dialyze the supernatant against 2x 1 liter Solution D.
• Quick freeze in liquid Nitrogen and store at -80
o.
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