Crude nuclear prep

Buffer A Crude Nuc Prep

Isolate nuclei first—use buffer A extraction – re-suspend cells in hypotonic solution without detergent so that the cells swell up, then add same buffer with detergent and they pop. Spin to collect nuclei, but save the cytoplasmic extract. Then re-suspend in nuclear lysis buffer (changes depending on experiment).


 * Trypsonize cells, quench with DMEM, spin down (as usual)


 * Aspirate ALL DMEM


 * re-suspend in 4 pellet volumes buffer A + dtt (1ul/mL) + pic- (20ul/mL)


 * DTT optional


 * For 3T3 cells: no dtt


 * Sit on ice 30 sec


 * For KCs Add equal volume buffer A + 0.4% igepal, and sit on ice 2 mins


 * For 3T3 or 293T cells: ½ volume!


 * Total volume should be 5-8 pellet volumes


 * Spin 1 min 4,000rpm 4C


 * Transfer sup to new tube (cytoplasmic fraction)—save for quant later


 * record amount of cyt extract removed


 * Spin again, 30 sec at 4,000 rpm to re-pellet nuclei


 * Use p20 to aspirate last cytoplasmic extract, toss. Keep nuc pellet on ice


 * Aspirate ALL cyt extract


 * Lyse nuclei with nuclear lysis buffer (keep concentrated and then dilute nuclei later)


 * Use 4-5 pellet volumes NLB


 * Re-suspend and then use 1-3mL syringe w/ 27.5 gauge needle to break up cells (push through needle a few times)


 * Leave on ice 30 mins (pipette or vortex to mix every 10mins)


 * Spin hard 5 mins (or 10mins) @ 4C @ max


 * Sup to new tube = nuc sup. Quant nuc sup and cyt extract with BCA kit


 * For KCs, ratio of protein yeilds should be 3 or 4 cyto: 1 nuc (protein content)


 * for 293Ts I beleive this is about 2:1 or 1:1

Buffer A —make 50mL @ RT…chill before use
 * 10mM Hepes pH 7.9 (0.5M stock = 500mM)
 * 1.5mM MgCl2 (1M stock = 1000mM)
 * 10mM KCl (2.5M stock = 2500mM)
 * In H2O


 * Add Pic- and DTT fresh (1mM)!!
 * Pic- @ 100X; DTT @ 1M
 * Pic- 10uL/mL; DTT 1uL/mL

50mL Buffer A:
 * 1mL 0.5M Hepes pH 7.9
 * 75uL 1M MgCl2
 * 200uL 2.5M KCl
 * + 48.73mL H2O
 * +PIC- & DTT!!!

Buffer A + 0.4% Igepal—make 50mL @ RT…chill before use
 * Buffer A + 0.4% Igepal (20% stock)
 * DON’T add Pic- or DTT (already in other buffer A, just dilute by 50% when add equal volume of Buffer A with detergent).

50mL BufferA + 0.4% igepal:
 * 1mL 0.5M Hepes pH 7.9
 * 75uL 1M MgCl2
 * 200uL 2.5M KCl
 * + 48.73mL H2O
 * 1mL 20% igepal

Nuclear lysis buffer for unfixed KCs—make 50mL and leave at 4C
 * 50mM Tris pH 7.6 (0.5M stock = 500mM)
 * 10% glycerol (100% stock)
 * 250mM NaCl (5M stock = 5,000mM)
 * 0.05% Igepal (20% stock)
 * Add Pic- fresh!! (@ 100X)

50mL Nuclear Lysis Buffer
 * 5mL 0.5M Tris pH 7.6
 * 5mL glycerol
 * 2.5mL 5M NaCl
 * 125uL 20% Igepal
 * + 37.375mL H2O
 * + PIC - !!!