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×Fixationsolution800ul使得甲醛的终浓度为1%，fixationsolution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1mlScellLysisBuffer，冰上放置10min，匀浆器匀浆后转入2mlEp管中，4℃5000RPM离心5min.5、弃上清，300ulnucleiLysisbuffer，吹散沉淀。6、socinate破碎：1minonoff3010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ChipDiluitonBuffer稀释样品10倍9、准备beads，用Pre-Blockingbufferfordynabeads洗beads3次.(ProteinA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10uldynabeads4℃颠转混匀2h。11、样品分成三组，A：加入trf2抗体5ul，B:加入Histone32ulC：不加任何抗体（A:B:C3:1:1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RNAPolymeraseII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10uldynabeads4℃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.lowsaltwashbuffer-onewash10minb.highsaltwashbuffer-onewash15minc.LiClwashbuffer-onewash30mind.TEbuffer2×5min14、洗涤完毕后加入150ulTE，加入pk液（10ul0.5MEDTA，20ul1MTris.HCl（PH6.5），1ul20mg/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。Histone4,、Ppp2r2c-its两对引物跑q-pcr。17、结果分析。1.Fixationsolution10mL11%formaldehyde(froma37%stockequilibratedwithmethanol)FinalconcentrationSourcevolume11%formaldehyde37%formaldehyde2.973ml100mMNaCl5MNaCl200ul1mMEDTA0.5MEDTA20ul0.5mMEGTA0.5MEGTA10ul50mMTRIS(pH8)1MTRIS500ulPrepareimmediatelybeforeuse.2.CellLysisbuffer(200ml)3.nucleiLysisbuffer(200ml)4.SDSLysisBuffer（100）5.ChipDiluitonBuffer(100mL)ddH2O6.3mlFinalconcentrationSourcevolume5mMPIPESPH8.00.5MPIPESPH8.02ml85mMKcl1MKcl17ml0.5%NP-40NP-401mlproteaseinhibitors50ul/mL现用现加ddH2O180mlFinalconcentrationsourcevolume1%SDS20%SDS10mlTris-cl（50mM）ph=8.01M10ml10mMEDTA0.5M4mlproteaseinhibitors50ul/mLddH2O176mlFinalconcentrationsourcevolume1%SDS20%SDS5mlTris-cl（50mM）ph=8.01M5ml10mMEDTA0.5M2mlddH2O88mlFinalconcentrationSourcevolume0.01%SDS20%SDS50ul1.1%TritonX-10010%TritonX-10011ml1.2mMEDTA0.5MEDTA240ul16.7mMTris1MTrisph=8.01.67ml167NaclNacl(5M)3.34mlddH2O83.7ml6.HighSaltWashBuffer（100ml）7.LowSaltWashBuffer（200ml）8.LiclWashBuffer（200ml）9.Phosphate-bufferedsaline(PBS)(for1X)Finalconcentration(10X)FinalconcentrationNaCl8g137mM80g1.37MKCl0.2g2.7mM2g27mMNa2HPO41.44g10mM14.4g100mMKH2PO40.24g1.8mM2.4g18mMFinalconcentrationSourcevolume2×TE10×TE20ml500mMNacl5MNacl10ml1%TritonX-10010%TritonX-10010ml0.1%SDS20%SDS500ulddH2O59.5mlFinalconcentrationSourcevolume2×TE10×TE40ml150MNacl5MNacl6ml1%TritonX-10010%TritonX-10020ml0.1%SDS20%SDS1mlddH2O133mlFinalconcentrationSourcevolume1×TE10×TE20ml0.25MLicl10MLicl5ml1%NP-40NP-402ml1%DOC10%DOC20mlddH2O153mlPBScanbemadeasa1Xsolutionorasa10Xstock.Toprepare1Lofeither1Xor10XPBS,dissolvethereagentslistedabovein800mLofH2O.AdjustthepHto7.4(or7.2,ifrequired)withHCl,andthenaddH2Oto1L.Dispensethesolutionintoaliquotsandsterilizethembyautoclavingfor20minat15psi(1.05kg/cm2)onliquidcycleorbyfiltersterilization.StorePBSatroomtemperature.10.10×TEbufferReagentQuantity(for100mL)FinalconcentrationsourcevolumeFinalconcentrationEDTA(0.5M,pH8.0)2mL10mMTris-Cl(1M,pH8.0)10mL100mMH2Oto100mL88ml11.Pre-Blockingbufferfordynabeads:1mL现配SDSLB95ulCHIPDB855ulbovineserumalbumin(BSA)40uL(5mg/mL)Yeast-tRNA10uL(20mg/mL)12.20%SDSAlsocalledsodiumdodecylsulfateorsodiumlaurylsulfate.Topreparea20%(w/v)solution,dissolve200gofelectrophoresis-gradeSDSin900mLofH2O.Heatto68°Candstirwithamagneticstirrertoassistdissolution.Ifnecessary,adjustthepHto7.2byaddingafewdropsofconcentratedHCl.Adjustthevolumeto1LwithH2O.Storeatroomtemperature.Sterilizationisnotnecessary.Donotautoclave.13.1MTris-ClTrisbaseHClTopreparea1Msolution,dissolve121.1gofTrisbasein800mLofH2O.AdjustthepHtothedesiredvaluebyaddingconcentratedHCl.pHHCl7.470mL7.660mL8.042mLAllowthesolutiontocooltoroomtemperaturebeforemakingfinaladjustmentstothepH.Adjustthevolumeofthesolutionto1LwithH2O.Dispenseintoaliquotsandsterilizebyautoclaving.Ifthe1Msolutionhasayellowcolor,discarditandobtainTrisofbetterquality.ThepHofTrissolutionsistemperature-dependentanddecreases~0.03pHunitsforeach1°Cincreaseintemperature.Forexample,a0.05MsolutionhaspHvaluesof9.5,8.9,and8.6at5°C,25°C,and37°C,respectively.14.0.5MEDTA(100ml)EDTA(ethylenediamenetetraaceticacidMW=372.24)NaOHToprepareEDTAat0.5M(pH8.0):Add18.61gofdisodiumEDTA•2H2Oto80mLofH2O.Stirvigorouslyonamagneticstirrer.AdjustthepHto8.0withNaOH(~20gofNaOHpellets).Dispenseintoaliquotsandsterilizebyautoclaving.ThedisodiumsaltofEDTAwillnotgointosolutionuntilthepHofthesolutionisadjustedto~8.0bytheadditionofNaOH,Adjusttotalvolumeto100mLwithH2O.15.1MKCl(Potassiumchloride)100mlFora1MsolutionofKCl,dissolve7.455gofKClin90mLofH2O.Makeupthevolum