《chip 操作步骤.docx》由会员分享，可在线阅读，更多相关《chip 操作步骤.docx（9页珍藏版）》请在三一办公上搜索。
1、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，加入11Fixation 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
2、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
3、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
4、与人和哺乳动物抗体的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 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10mi
5、n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 25min 14、洗涤完毕后加入150ulTE，加入pk液，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-pc
6、r。 17、结果分析。 1. Fixation solution 10mL 11% formaldehyde (from a 37% stock equilibrated with methanol) Final concentration Source volume 11% formaldehyde 100 mM NaCl 1 mM EDTA 0.5 mM EGTA 50 mM TRIS (pH 8) 37% formaldehyde 2.973ml 5M NaCl 200ul 0.5M EDTA 20ul 0.5M EGTA 10ul 1M TRIS 500ul ddH2O 6.3ml P
7、repare immediately before use. 2. Cell Lysis buffer(200ml) Source volume Final concentration 5mM PIPES PH 8.0 85mM Kcl 0.5% NP-40 protease inhibitors ddH2O 0.5M PIPES PH 8.0 2ml 1M Kcl 17ml NP-40 1ml 50ul/mL 现用现加 180ml 3. nuclei Lysis buffer(200ml) Final concentration source 1%SDS 20%SDS Tris-cl ph=
8、8.0 1M 10mM EDTA 0.5 M protease inhibitors ddH2O 4. SDS Lysis Buffer Final concentration source 1%SDS 20%SDS Tris-cl ph=8.0 1M 10mM EDTA 0.5 M ddH2O volume 10ml 10ml 4ml 50ul/mL 176ml volume 5ml 5ml 2ml 88ml 5. Chip Diluiton Buffer(100mL) Source volume Final concentration 0.01%SDS 1.1%Triton X-100 1
9、.2 mM EDTA 16.7mM Tris 167 Nacl ddH2O 20%SDS 50ul 10%Triton X-100 11ml 0.5M EDTA 240ul 1M Tris ph=8.0 1.67ml Nacl(5M) 3.34ml 83.7ml 6. High Salt Wash Buffer Source volume Final concentration 2TE 500mM Nacl 1%Triton X-100 0.1%SDS ddH2O 10TE 20ml 5M Nacl 10ml 10%Triton X-100 10ml 20%SDS 500ul 59.5ml 7
10、. Low Salt Wash Buffer Source volume Final concentration 2TE 10TE 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 Source volume Final concentration 1TE 10TE 20ml 0.25M Licl 10M Licl 5ml 1%NP-40 NP-40 2ml 1%DOC 10%DOC 20ml ddH2O 153ml
11、9. Phosphate-buffered saline (PBS) (for 1X) Final concentration NaCl 8 g KCl 0.2 g Na2HPO4 1.44 g KH2PO4 0.24 g 137 mM 2.7 mM 10 mM 1.8 mM (10X) 80 g 2 g 14.4 g 2.4 g Final concentration 1.37 M 27 mM 100 mM 18 mM PBS can be made as a 1X solution or as a 10X stock. To prepare 1 L of either 1X or 10X
12、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 ro
13、om temperature. 10. 10TE buffer Reagent Quantity (for 100 mL) Final concentration source EDTA (0.5 M, pH 8.0) Tris-Cl (1 M, pH 8.0) H2O to 100 mL volume 2 mL 10 mL 88ml Final concentration 10 mM 100 mM 11. Pre-Blocking buffer for dynabeads: 1mL 现配 SDS LB 95ul CHIP DB 855ul bovine serum albumin(BSA)
14、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 68C and stir with a magnetic stirrer to assist dissolution. If necessary, adjust the
15、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 va
16、lue by adding concentrated HCl. pH HCl 7.4 70 mL 7.6 60 mL 8.0 42 mL Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 L with H2O. Dispense into aliquots and sterilize by autoclaving. If the 1 M solution has a yellow colo
17、r, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases 0.03 pH units for each 1C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5C, 25C, and 37C, respectively. 14. 0.5M EDTA(100ml) EDTA (ethylene
18、diamenetetraacetic acid MW=372.24) NaOH To prepare EDTA at 0.5 M (pH 8.0): Add 18.61 g of disodium EDTA2H2O to 80 mL of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA wi
19、ll not go into solution until the pH of the solution is adjusted to 8.0 by the addition of NaOH, Adjust total volume to 100 mL with H2O. 15. 1M KCl (Potassium chloride)100ml For a 1 M solution of KCl, dissolve 7.455 g of KCl in 90 mL of H2O. Make up the volume to 1ooml with H2O and autoclave for 20
20、min on liquid cycle. Store at room temperature. Ideally, this solution should be divided into small (100 L) aliquots in sterile tubes and each aliquot thereafter used only once. 16. 5 M NaCl (Sodium chloride)100ml To prepare a 5 M solution: Dissolve 29.2 g of NaCl in 80 mL of H2O. Adjust the volume
21、to 100 ml with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at room temperature. 17. 1M EGTA (Ethylene glycol-bis-aminoethyl ether-N,N,N,N-tetraacetic acid) (100 ml) EGTA (m.w. = 380.4) Add 25g of EGTA to 50 mL H2O. Adjust the pH to 7.0 with HCl or Nacl. Adjust t
22、otal volume to 65.72 mL with H2O. Filter, autoclave, and store at 4C (lasts 1 mol). 18. 10 M LiCl 250ml To prepare 4 M lithium chloride: Dissolve 42.4 g of LiCl in a final volume of 200 ml of H2O. Adjust the volume of the solution to 250 ml with H2O. Sterilize the solution by passing it through a 0.
23、22-m filter, or by autoclaving for 15 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. Store the solution at 4C. 19. 1 M glycine (FW = 75.07) 3.754 g / 50 mL ddH2O filtrate and store at RT 20. 1M PIPES (10ml) 1. 25g PIPES(MW= 302.37) 2. Dissolve PIPES in a small volume of H2O. 3. Adjust the final v
24、olume to 82.68mL with ddH2O. 4. Filter through a 0.45-m sterile filter. 5. Store in the dark at 4C or aliquot and freeze. (The buffer is stable for 2 mo at 4C). Employ aseptic technique when diluting the PIPES buffer for preparation of 1X working solutions or store as single-use aliquots to avoid contamination. 21. NaCl (Sodium chloride) To prepare a 5 M solution: Dissolve 73.05g of NaCl in 150 mL of H2O. Adjust the volume to 500 mL with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at room temperature.
