chip 操作步骤

磁珠吸附法chip操作步骤及试剂配方 1、每盘细胞（8ml培液），加入11×Fixation solution 800ul使得甲醛的终浓度为1%，fixation solution为现配，室温摇床10min。(甲醛能有效的使蛋白质-蛋白质，蛋白质-DNA，蛋白质-RNA交联，形成生物复合体，防止细胞内组分的重新分布。甲醛的交联反应是完全可逆的，便于在后续步骤中对DNA和蛋白质进行分析; 交联时间如果过长，细胞染色质难以用超声波破碎，影响ChIP结果，而且实验材料也容易在离心过程中丢失。交联时间如果过短，则交联不完全，产生假阴性。甲醛的交联反应可被加入的甘氨酸终止。) 2、终止交联：加1M甘氨酸1.26ml，使其终浓度为0.125。室温摇床5min。 3、用冰冷的PBS 冲洗两次后，加适量pbs+pmsf，用细胞刷刮下细胞，4℃ 1000RPM离心5min。 4、倒去上清，加入1ml Scell Lysis Buffer，冰上放置10min，匀浆器匀浆后转入2ml Ep管中，4℃ 5000RPM离心5min. 5、弃上清，300ul nuclei Lysis buffer，吹散沉淀。 6、socinate破碎：1min on  off 30 10次左右，4℃ 13000RPM离心20min，琼脂糖胶电泳，亮带集中在1000bp左右的方可。  (以便暴露目标蛋白，利于抗体识别。) 7、分别收集20ul样品做input -20℃保存。(Input是断裂后的基因组DNA，需要与沉淀后的样品DNA一起经过逆转交联，DNA纯化，以及最后的PCR或其他方法检测。Input对照不仅可以验证染色质断裂的效果，还可以根据Input中的靶序列的含量以及染色质沉淀中的靶序列的含量，按照取样比例换算出ChIP的效率，所以Input对照是ChIP实验必不可少的步骤。) 8、用 Chip Diluiton Buffer 稀释样品10倍 9、准备beads，用Pre-Blocking buffer for dynabeads 洗beads3次.( Protein A是一种金黄色葡萄球菌细胞壁蛋白质，能特异性地与人和哺乳动物抗体（主要是IgG）的Fc区结合)，最好实验前一天准备beads。 10、样品中各加入10ul dynabeads 4℃颠转混匀2h。 11、样品分成三组，A：加入trf2抗体5ul，B:加入Histone3 2ul C：不加任何抗体（A:B:C 3:1:1的体积）4℃过夜。(阳性抗体通常选择与已知序列相结合的比较保守的蛋白的抗体，常用的包括组蛋白抗体或RNA Polymerase II抗体等。阴性抗体通常选择目的蛋白抗体宿主的IgG或血清) 12、分别向A、B、C各样品加入10ul dynabeads 4℃颠转混匀2h。 13、 洗涤beads（在4℃冷库操作比较方便 dynamag把磁珠洗住，就可以把洗涤液洗掉，最好使用枪头吸不要嫌麻烦） 依次用下列溶液清洗沉淀复合物。清洗的步骤：加入溶液，在4℃颠转10min，4℃静置10min沉淀，700rpm离心1min，除去上清。 洗涤溶液：a.low salt wash buffer-one wash 10min b.highsalt wash buffer-one wash 15min c.LiCl wash buffer-one wash 30min d.TE buffer  2×5min 14、洗涤完毕后加入150ulTE，加入pk液（10ul 0.5MEDTA，20ul 1MTris.HCl（PH6.5），1ul 20mg/ml蛋白酶K）45℃颠转2h，input也要进行此步。 15、去除磁珠，收集样品液，DNA试剂盒纯化dnA,检测od值。 16、realtime-pcr分析样品。Histone 4,、Ppp2r2c-its两对引物跑q-pcr。 17、结果分析。 1. Fixation solution  10mL 11% formaldehyde (from a 37% stock equilibrated with methanol)                       Final concentration Source volume 11% formaldehyde 37% formaldehyde 2.973ml 100 mM NaCl 5M NaCl 200ul 1 mM EDTA 0.5M EDTA 20ul 0.5 mM EGTA 0.5M EGTA 10ul 50 mM TRIS (pH 8) 1M TRIS 500ul ddH2O 6.3ml     Prepare immediately before use. 2. Cell Lysis buffer(200ml) Final concentration Source volume 5mM PIPES PH 8.0 0.5M PIPES PH 8.0 2ml 85mM Kcl 1M Kcl 17ml 0.5% NP-40 NP-40 1ml protease inhibitors 50ul/mL 现用现加 ddH2O 180ml     3. nuclei Lysis buffer(200ml) Final concentration source volume 1%SDS 20%SDS 10ml Tris-cl（50mM） ph=8.0 1M 10ml 10mM EDTA 0.5 M 4ml protease inhibitors   50ul/mL ddH2O   176ml       4. SDS Lysis Buffer（100） Final concentration source volume 1%SDS 20%SDS 5ml Tris-cl（50mM） ph=8.0 1M 5ml 10mM EDTA 0.5 M 2ml ddH2O   88ml       5. Chip Diluiton Buffer(100mL) Final concentration Source volume 0.01%SDS 20%SDS 50ul 1.1%Triton X-100 10%Triton X-100 11ml 1.2 mM EDTA 0.5M EDTA 240ul 16.7mM Tris 1M Tris ph=8.0 1.67ml 167 Nacl Nacl(5M) 3.34ml ddH2O 83.7ml     6. High Salt Wash Buffer（100ml） Final concentration Source volume 2×TE 10×TE 20ml 500mM Nacl 5M Nacl 10ml 1%Triton X-100 10%Triton X-100 10ml 0.1%SDS 20%SDS 500ul ddH2O 59.5ml     7. Low Salt Wash Buffer（200ml） Final concentration Source volume 2×TE 10×TE 40ml 150M Nacl 5M Nacl 6ml 1%Triton X-100 10%Triton X-100 20ml 0.1%SDS 20%SDS 1ml ddH2O 133ml     8. Licl Wash Buffer（200ml） Final concentration Source volume 1×TE 10×TE 20ml 0.25M Licl 10M Licl 5ml 1%NP-40 NP-40 2ml 1%DOC 10%DOC 20ml ddH2O 153ml     9. Phosphate-buffered saline (PBS) (for 1X) Final concentration (10X) Final concentration NaCl 8 g 137 mM 80 g 1.37 M KCl 0.2 g 2.7 mM 2 g 27 mM Na2HPO4 1.44 g 10 mM 14.4 g 100 mM KH2PO4 0.24 g 1.8 mM 2.4 g 18 mM         PBS can be made as a 1X solution or as a 10X stock. To prepare 1 L of either 1X or 10X PBS, dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.  10. 10×TE buffer Reagent Quantity (for 100 mL) Final concentration source volume Final concentration EDTA (0.5 M, pH 8.0) 2 mL 10 mM Tris-Cl (1 M, pH 8.0) 10 mL 100 mM H2O to 100 mL 88ml         11. Pre-Blocking buffer for dynabeads:    1mL  现配 SDS LB 95ul                  CHIP DB 855ul bovine serum albumin(BSA)      40uL (5mg/mL) Yeast-tRNA                    10uL (20mg/mL) 12. 20%SDS Also called sodium dodecyl sulfate or sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of electrophoresis-grade SDS in 900 mL of H2O. Heat to 68°C and stir with a magnetic stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of concentrated HCl. Adjust the volume to 1 L with H2O. Store at room temperature. Sterilization is not necessary. Do not autoclave. 13. 1M Tris-Cl Tris base HCl To prepare a 1 M solution, dissolve 121.1 g of Tris base in 800 mL of H2O. Adjust the pH to the desired value by adding concentrated HCl.