109分子生物学实验技术简册（PDF56页）

DNADNARNAcDNADNARFLPRAPD(PCR)DNADNADNA,(Vector)DNA(Plasmid),1-200kb,DNA,,,,,F(F)R()Col():(Stringentcontrol)(Relaxedcontrol),,,1,F,,20,ColE1-,DNA,,,201000-3000,DNADNA2%40-50%,,,,,,,(Incompatibility),,,,,(),,,,DNADNA123DNA41kb10kbPBR322PUCPGEMpBluescriptpBSDNA3:;DNA,(SDS)TritonX-100SDSTritonX-100,DNA,DNA,DNA,DNA(CovalentlyclosedcircularDNAcccDNA)DNA,,DNA,,,DNADNADNA,,,DNA(OpencircularDNA,ocDNA)DNA,DNA(LinearDNA)DNA,DNApBSE.coliDH5JM1.5ml(eppendorf),(20l,200l,1000l)()1LB(Luria-Bertani)(Tryptone)10g(Yeastextract)5gNaCl10g800mlNaOHpH7.5,1,202LB12g,3(Ampicillin,Amp)50mg/ml,-20410mmol/LTrisCl(pH8.0)10mg/ml,(1.5ml)-20,53mol/lNaAc(pH5.2)50ml40.81gNaAc3H2O,pH5.2,100ml,,4650mmol/L25mmol/LTris.Cl(pH8.0)10mmol/LEDTA(pH8.0),100ml,15,470.2mol/LNaOH(10mol/LNaOH)1SDS85mol/LKAc60ml11.5mlH2O28.5ml100ml,:K+3mol/L,Ac5mol/L9RNAARNAA10mmol/LTrisCl(pH7.5),15mmol/LNaCl,10mg/ml,10015,DNA1.5mleppendorf-2010,RNADNA,1600.18-(),0.5mol/LTrisCl(pH8.0)0.1mol/LTrisCl(pH8.0)pH7.6,DNA11::24:1,/1:1/(1:1)12TE10mmo/LTrisCl(pH8.0)1mmol/LEDTA(pH8.0)413STET0.1mol/LNaCl10mmol/LTrisCl(pH8.0)10mmol/LEDTA(pH8.0)5%TritonX-10014STE0.1mol/LNaCl10mmol/LTrisCl(pH8.0)1mmol/LEDTA(pH8.0)151TBE(5)Tris54g27.5g0.5MEDTA(pH8.0)20ml1000ml2(6)0.25%40%(w/v)pBSDH5LB(50g/mlAmp),3712-245mlLB(50g/mlAmp),3712DNADNADNA,DNA,11.5mleppendorf,4120003023120mlSTET,410ml(10mg/ml),35eppendorf,506412000g107eppendorf820ml[]1.DNA2.,,3.DNARNA,RNA,,11.5ml1.5mleppendorf,412000g302,,3100l(),5-104200l,eppendorf,(),55150l,10,,5-10,412000g5-106eppendorf,/(1:1),,412000g57eppendorf,2,-2020412000g108,,1ml70,412000g5-109,,101020lTE(pH8.020g/mlRNaseA)-20[]1.2.DNA,/,3.DNA,DNADNA(),,,,WizardDNAPromegaWizardDNADNA,15DNA501-3ml10ml10ml10ml50mlWizardDNA50ml95%120ml)50Wizard11-3ml412000g1-22200l,,eppendorf3200l,,4200l,5412000g5eppendorf61mlWizardDNA,7,,Wizard,,,8,,,2ml,9eppendorf,210eppendorf50lTE()1412000g2011eppendorfDNA4-20[],,25-3710DNADNA,DNADNA,1pBS250ml45000g15,,250mlSTE3145mlI,512mlII,,,,1069mlIII,,15,745000g158,50mlRNAA(10mg/ml),37209/,,412000g1010,,,412000g1011,1/54mol/LNaCl10%PEG(6000),6012412000g15,ml70%412000g513500mlTE[]1.2.IIIII,3.RNAADNA,RNA,(801),DNAWazardDNADNA.PromegaWiazrdDNA,,500ml31mgDNA(200-20000bp),DNATEDNARNasin10100-500ml,:150ml150ml150ml100mlWizardDNA125mlWizard10Wizard1100-500ml,22-255000g10,215ml,,,,,20315ml,414000g,22-2515560.5,,14000g22-25157,DNA2mlTE810mlWizardDNA,9,Wizard,(Promega,)10/DNA,/DNA11/DNA,13ml,/DNA()121312ml145ml80,,2500rpm(1300g)5155,2500rpm(1300g)5161.5ml65-70TE,12500rpm(1300g)/517,DNA,,4-20[]1.,1:1125ml952..Sephrose2BDNADNARNA,RNARNADNA,Sepharose2BSepharose4B,,,,,DNA1Sepharose2B0.1%SDSTEpH8.021mlDNASepharase2B3DNA0.1%SDSTE(pH8.0)41ml15OD260,DNADNA6,/(1:1),412000g2,72-2010,412000g10,870%,412000g10,9,TE[],,,0.1%SDSTE,1.2.3.DNADNA.DNADNA,DNA:()ATPDNADNA,:(),;,DNA46(EcoR:5'-GAATTC-3'),,,DNA(Sma:5'-CCCGGG-3');,DNA,EcoR5'GAATTC3'5'GAATTC3'3'CTTAAG5'3'CTTAAG5'DNARNADNA,,,,,,,,/0.13mol/LNaAc2-7030DNADNADNADNARFLP()DNADNA0.5-1g,(37)DNA,1gDNA1,1-2,2-3,,.DNADNA(Ethidiumbromide,E,1-10ngDNA,DNA,DNA,DNA,200bp50kbDNADNA(5-500bp),,1bpDNA,DNA,,DNADNA,,pHDNA,:1DNA:DNADNA2DNA,DNADNA0.5kbDNA1.2-1.5%,10kbDNA0.3-0.7%,DNA0.8-1.0%3DNADNA,,DNA,DNA,DNADNADNADNA,DNA,,,DNA,DNA4,DNADNA,,,2kbDNA,5v/cm5DNA,DNA,DNA156DNA(),,DNA,(10),,DNADNA,TAE[EDTA(pH8.0)Tris-],TBE(Tris-EDTA),TPE(Tris-EDTA),,DNA:;pBSpUC19;EcoRI:;Hind:;(Agarose):,,,,,,,,15TBE260.2540(w/v)43(E:EB10mg/ml,,DNA1eppendorf(0.5ml),DNA1g102l,19l,1l,,,,2,eppendorf,372-3,32l0.1mol/LEDTA(pH8.0),,,DNAEcoRHindDNADNA50kbDNA,0.5mg/ml,HindIIIDNA8,23.1,9.4,6.6,4.4,2.3,2.0,0.560.12kbEcoRIlDNA6,21.2,7.4,5.8,5.6,4.92.5kb15TBE20ml200ml,0.5TBE,20.4g,200ml,50ml0.5TBE,(),,0.8,,3(1cm),,1mm50-60(E0.5g/ml(EB,0.5g/mlEB),,,,,,,0.5TBE,,410l2l6,DNA,,tip,,,5,,60-80V,40mA2cm,6EB0.5g/mlEB,20-257254nmEBDNA,5.6,10-120()8DNA,,DNAEcoRHind,,,,DNA9DNA,DNA,,DNA,(,DNA),DNA10DNA,,DNA,,DNA[]1.DNADNA2U11mglDNADNAlDNA3150%1/104DNA,DNADNA,(300-360nm,DNA5EB,,EBEB6EB,,DNA,,301.DNADNA,2.DNA,,,mob,DNA(Transformation)DNA,,(R,M),DNA(,CaCl2,RbCl(KCl)),,DNA(Compenentcells)DNA,,(Transformant,DNA)CaCl2RbCl(KCl)RbCl(KCl),CaCl2,,,15-70(),CaCl2,1.:70-20OD600DH5OD6000.5,5107/ml(),2.:DNADNA(cccDNA)DNA,DNA,1ngcccDNA50l,DNA53.:,CaCl2(GR.AR.),,4.DNA,,,tip,,,DNADNA,DNA,E.coliDH5a,CaCl2,,pBS,pBS(Ampr)AmppBSAmpAmppBSE.coliDH5a,CaCl2,,pBS,pBS(Ampr)AmppBSAmpAmppBS.E.coliDH5:R,M,AmppBSDNA:eppendorf.,,,,,,,,.1.LB2.Amp3.AmpLB:LB60,Amp,50ug/ml,4.(MaconkeyAgar):52g,1000ml,,60Amp50ug/ml,5.0.05mol/LCaCl2:0.28gCaCl2(,),50ml,100ml,6.15%0.05mol/LCaCl2:0.28gCaCl2(,),50ml,15ml,100ml,LBE.coliDH5,3-5mlLB,3712,1:100-1:50100mlLB,372-3OD6000.5(CaCl2)1,10,43000g102,0.05mol/LCaCl210ml,15-30,43000g103,4ml15%0.05mol/LCaCl2,,,4200l,-701-70200l,,2pBSDNA(50ng,10l),,3034290375,3-541mlLB(Amp),371,,(Ampr)5100lAmp,,,3716-24:1:DNA,LB2:DNA,5lLBmgDNADNA(mg)[]E.coliDNA,,(),1.2.RNAcDNAmRNA,cDNA,cDNA,,cDNA,cDNA;cDNADNAcDNA70cDNA,cDNA,cDNAcDNAcDNA,cDNA,DNA()RNAmRNAcDNAmRNA,RNA,RNA,RNA,,RNA,20020.1%(DEPC),DEPCRNA,RNADEPC,DEPC,DEPC0.1%,10,15DEPC,DEPCmRNADEPC,TrisDTTDEPCTrisDEPC,DEPC,DEPC,RNA,mRNAcDNA,RNA,RNA,RNARNAmRNA3'(A)+,