分子生物学实验技术

...............................................................................................1DNA...............................................................4DNA....................................................7.................................................................................11.................................................................14PCR...............................................................................................18DNA............................................................20DNA....................................................................23Southern.....................................................................26Southern.....................................................................30RNA..........................................................................38RT-PCR.......................................................................................40RNA..............................................................4313DNApUC18/19BAC1.5mlDNADNApH12.012.6DNADNApHDNARNASDSDNADNARNADNADNA11ml410000r/minmin,,2150LI,10min23200LII,,,,5min4150LIII,,15min,5410000r/min15min6,:,,410000r/min10722min5min81mL70%pH8.0TE-20pUC192686bpAPrALPHAP(BLA)P(LAC)ORIAvaI(413)BamHI(418)EcoRI(397)HindIII(448)PstI(440)SmaI(415)XmaI(413)ApaLI(178)ApaLI(1121)ApaLI(2367)3260nm280nm260nm/280nm1.71.91.81.81.8RNA1.EDTASDS2.3.4.4DNADNApH8.08.3EB1230.24g30ml0.5TBE45-5045pUC185l+ddH2O3l+2l10l0.5mltube5670.5g/mlEB1015min1DNADNAUlogNNDNADNAlogU=logU0KrtUU0DNAtKrDNAAgarose0.5%1-30kb0.7%0.8-12kb1.2%0.4-7kb1.5%0.2-3kb.DNADNAEBDNA5V/cm4-30DNA()60.5TBEDNADNA1TAE1TBE1TPEEDTApH8.02EB3bromophenolblue,Bbxylenecyanol,Xc1DNA2bpDNA?30.15kb0.2kb0.6kb1kb21.410.6%1.6kb2kb7DNADNADNADNADNABACDNApUC18DNABACPUC18DNADNApUC181pUC18DNA50l1.5mltubeDNA30lR.E1l10buffer5lddH2O14l8371hr8l5lPUC181.0%25DNA2ddH2O150l/24:112000g10min31/103MNaAc-2015412000g415575DNA10lddH2O0.5mltubePUC1820lddH2O1.5mltube6PUC185'5030minDNA20lCIAPTaKaRa0.5l10buffer4.0lddH2O15.5l77010min,CIAP82510lddH2O915lDNA10lpUC182.5l5buffer1.5lT4ligase(3U/l)1l16101191211U50l3711gDNAstarDNADNAstarDNA2BAPCIAPDNARNAdNTPNTP5'CIAP7010'CIAPBAP102015'-P2Kinase5'DNA5'-P3T4T410pUC192686bpAPrALPHAP(BLA)P(LAC)ORIAvaI(413)BamHI(418)EcoRI(397)HindIII(448)PstI(440)SmaI(415)XmaI(413)ApaLI(178)ApaLI(1121)ApaLI(2367)pUC192686bpAPrALPHAP(BLA)P(LAC)ORIpUC193237bpAPrP(BLA)P(LAC)ORIAvaI(413)EcoRI(397)SmaI(415)XmaI(413)ApaLI(178)ApaLI(1672)ApaLI(2918)ClaI(869)ClaI(897)ClaI(996)+A11DNADNACaCl2DNACaCl2DH5DH10B1091010/gDNACaCl2DNA106107/gDNACaCl21DH5DH10BLB371621ml100mlLB372.5-3250-300rpm30.1MCaCl241.5ml1.5ml1012543000g56100l0.1MCaCl220743000g58100l0.1MCaCl2915%-20%701DH5DH10BLB3722ml200mlLB37OD600=0.62.5-3341.5ml1.5ml10543000g561500l10%743000g58750l10%13943000g51020l10%11-701OD600DH5OD6000.55107/ml23CaCl2244Ca2+Mn2+Co2+DMSO100-100056DNA7DNA1410CaCl2DNADNase42DNA2DNADNA1250mlLA250lAmp100mg/ml250lX-gal(20mg/ml)25lIPTG(200mg/ml)23153100l20ngDNA3DNADNA30442905126400lSOC37457IPTGX-galAmpLA83712161250mlLA250lAmp100mg/ml250lX-gal(20mg/ml)25lIPTG(200mg/ml)231l41800V51mm61mlSOC1673718937190mm10537202DNApBR322;PCR1.2.17PCRpolymerasechainreaction,PCRPCRPCRPCRDNAPCRDNAdNTPsDNAPCRDNA15ng/l2PCR5TemplateDNA2l20ng10buffer2.0lMgCl225mM1.5lPrimerF(10M)0.2lPrimerR(10M)0.2ldNTPs2mM2.0lTaq5U/l0.2lAddddH2Oto11.9l3PCR953'1cycle941'571'721'3035cycles18728'1cycle4forever42l15l51%EB12PCR3TmPCRPCR4PCRDNADNA19DNADNADNADNACTABDNA1.5CTAB/(24:1)95%DNA1SDSSDSKDNA2CTABDNA()CTABCTAB65DNACTAB0.7mM0.1-0.5mMNaClCTAB-CTAB-7075%CTAB/(24:1)DNADNADNA201N20.4g1.5ml-2021.5CTAB951ml36530min5min1412000g5min5600l600l/(24:1)612000g57450l1.5ml1ml95%45l10MNH4AC10min812000g10min75%EtOH30min950l1/10TE20g/RNase4DNADNA110mM-ME2CTAB324:1/45260nm280nm230nmDNAOD260/OD2801.7OD260/OD2302.0211DNA2DNA3DNA22DNADNADNADNADNADNADNA,DNA:()ATPDNADNA,:(),;,DNA48DNADraIEcoRIEcoRVHindIII110lDNA0.8%2DNA300-400ng/l310U-50U/l40.5mltube23DNA(3-5g)10l10bufferreaction1.5lEnzyme(15U/l)0.8lddH2O2.7l5371-2hrs(DNA)10hrsDNA66510min71/10DNADNADNADNADNA1.2.3.EB244.EcoRIHind5.6.25SouthernDNARFLPSouthernblotting12DNA500bpDNADNADNADNA1010-20DNADNA80-1002-4hrsDNA263transferbufferSSC0.4NNaOHDNADNA20SSCDNApH9DNA4durationoftransfer12hrsDNA(5mm)(1%)DNADNA2hrsDNAEBDNA0.2NHCl0.4NNaOH10.8%5mm250ml0.8%42DNA1-1.5V/cmDNA10-11cm(40V12-15hrs30V4-5hrs)2271112.5cm1010.5cm30.4NNaOH()4a.b.0.2NHCl10minc.HCld.0.4NNaOHe.0.4NaOH560.4NNaOH0.4NNaOH721512cm85cm500g12hrs92SSC5minEB1080-1002-4hrs281229SouthernDNAEBDNAXDNADNADNADNADNADNA1DNADNADNADNA2DNARNAnicktranslationinvitrotranscriptionPCR5'3'DNADNAheterogeneous646=409630DNATheklenowfragmentE.coliDNApolymeraseI5'3'5'3'3'5'PrimerDNaseIDNADNADNA[=k/(lnPc)1/2Pc]400-600bpDNADNADNADNAdNTPs3DNA4DNA32PdCTP20SSC10%SDSX-12SSC653hrs6-12hrs2311-2blots(19.59.5cm2)DNA100ngDntp2.0lRandomprimer5.0lKlenow(1u/l)1l-dCTP*1.0laddddH2Oto17.0lDNA1.510010min5mindNTPRandomprimerKlenowDNA32PdNTP-32PdCTP*3000Ci/mmol303hrs3300l100or0.4NNaOH25%41SSC/0.1%SDS5min6515min0.5SSC/0.1%SDS6515min0.2SSC/0.1%SDSor0.1SSC/0.1%SDS325X--20-7