chip protocol实验室成熟的chip-seq及chip-qpcr操作流程
3页1、ChIP protocol Fixation type: Type I:1% formaldehyde /PBS(100ml): 36.5%formaldehyde (F8775-500ml) 2.5ml diluted in 1XPBS 88.75 mlRoom temperature, 15minType II:2-5mM DSG (stock 50mM: 10mg in 540ul of dry DMSO), Room temperature, 45min1% formaldehyde /PBS, Room temperature, 15minType III:1% Glutaraldehyde solution(Grade I, 25% in H2O, specially purified for use as an electron microscopy fixative) sigma G5882-100ml, Room temperature, 15minStock buffer: 20%SDS; 0.5M, pH8.0 EDTA; 1M, pH7.8 Tris-Cl; 1
2、0% TritonX100; 5M NaCl, Buffer recipe:Lysis Buffer10ml2ml20%SDS0.5ml100ul0.5M, pH8.0 EDTA0.2ml50ul1M, pH7.8 Tris-Cl0.5ml100ul50Xcocktail(Roche)0.2ml(add freshly)40ul(add freshly)Double Distilled water8.6ml1720ulDilution Buffer500ml100ml10% TritonX10050ml10ml0.5M, pH8.0 EDTA2ml0.4ml5M NaCl15ml3ml1M, pH7.8 Tris-Cl10ml2ml50Xcocktail(Roche)10ml(add freshly)2ml(add freshly)Double Distilled water413ml82.6ml100mlTSE1TSE220%SDS0.5ml0.5ml10% TritonX10010ml10ml0.5M, pH8.0 EDTA0.4ml0.4ml5M NaCl3ml8ml1M, pH
3、7.8 Tris-Cl2ml2ml50Xcocktail(Roche)1ml(add freshly)1ml(add freshly)Double Distilled water83.1ml78.1ml1.25M Glycine(MW: 75.07): 18.77g plus 200ml ddw 5M NaCl(MW58.45): 292.2g plus 1L ddw, autoclave20% SDS(MW288.38): 20g plus 100ml ddw1M, pH7.8 Tris-Cl: 121.1g plus 1L ddw, autoclaveOption: Drug treatment:10-7 M E2 treat 1hrDay1: cell fixation, cell lysis, sonication and antibody incubationCollecting cells1. After fixation, cold PBS wash twice2. 1ml PBS to scrape cells and 0.5ml PBS scrape cells 3.
4、 Transfer cells into 1.5ml tubes, max speed 30sec1min, centrifugation at 44. If you want to stop at step3, you can freeze the samples by liquid nitrogen quickly, then store at -80 for long time storage. If not, continue to step 5Cell lysis and sonication5. Add 300ul lysis buffer plus complete cocktail (avoid creating bubbles)6. Sonication(声波降解法): we use bioruptor for sonication: 30sec/30sec, the cycles of sonication dependent on cell type, cell numbers and crosslinking methods. (for example: 293
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