DNA技术课件

《DNA技术课件》由会员分享，可在线阅读，更多相关《DNA技术课件（36页珍藏版）》请在装配图网上搜索。

1、免疫学信息网 http:/Techniques to manipulate DNAPhenol/chloroform extractionEthanol precipitationConcentration determinationGel ElectrophoresisDNA hybridizationSouthern BlottingMelting temperaturesPCR免疫学信息网 http:/BRCA3EcoRIEcoRINIHExample:Is the BRCA3 gene involved in Suppressing breast cancer?BRCA1BRCA2BR

2、CA3免疫学信息网 http:/Paper TowelsSouthern Blotting1tissueSDSProtKPhenolChloroETOH pptSpin免疫学信息网 http:/The“Hyperchromic Shift”2免疫学信息网 http:/Paper TowelsSouthern Blotting1tissueSDSProtKPhenolChloroETOH pptSpin免疫学信息网 http:/Agarose Gel Electrophoresis_+3免疫学信息网 http:/Sample Well 25 ng 1 kb ladder0.8%Agarose12

3、4681012kbAgarose Gel ElectrophoresisSybergold Detection Limit:0.2-0.5 ng DNAEtBr Detection Limit:5-10 ng DNA(Not in handout)免疫学信息网 http:/Log(MW)is linearly related to mobility4100 bpLow%has noResolving powerat low MW1 kb10 kbHigh%has noresolving powerat high MW免疫学信息网 http:/Separation Range Vs.%Agaro

4、se5Low Geling Agarose 4-6 0.02-0.4免疫学信息网 http:/TEMED(N,N,N,N-tetramethylenediamine)7免疫学信息网 http:/PolyAcrylamide Gels8免疫学信息网 http:/DNA topology affects mobility of DNA 9免疫学信息网 http:/PFGE can resolve large DNA fragments10免疫学信息网 http:/Paper TowelsSouthern Blotting1tissueSDSProtKPhenolChloroETOH pptSpin

5、免疫学信息网 http:/Helix Formation:Nucleation and Propagation11免疫学信息网 http:/A-TA-TG-CHelix Melting12免疫学信息网 http:/What parameters affect theTm?(Tm is defined as the temp at which half the moleculesare single-stranded)免疫学信息网 http:/Tm depends on DNA length13%dsDNASigmoidalCurve:HighlyCooperativeMelting!免疫学信息网 http:/Effect of%GC Content on Tm14免疫学信息网 http:/Effect of Salt on Tm15免疫学信息网 http:/i)For oligos 4-18 nucleotidesTm=(#A-T bp)2 C +(#G-C bp)4Cii)For 14 70 nucleotides0.01 M Na+0.4 M,and 30%GC 2 kbrequires low voltage0.5 10 V/cm6