PCRED 采集土样(3个重复,0.5g DNA提取( DNA SPI试剂盒 [6FAMJA GA GTTTGATCCTGGCTCAG- ATP TTP FAST DNA PREI 3)和反向引物38R( TP. CTP YSTEM清除蛋白质和其他杂质 )获得DNA模板 GCTGCCTCCCGTA GGA GT-3)合成 各02mM 10 0.06%牛血清白蛋白,1.5 mM MgCl2,2单位Taq DNA聚合酶,1×PCR缓冲液 9℃初始变性3分钟 55℃退火45秒 94℃变性45秒 72℃延长2分钟 建立系统树 72℃保持7分钟 取 IngPCR产物 宽区聚丙烯酰氨凝胶电泳 聚丙烯酰氨变性梯度凝胶电泳 条带分析
采集土样（3个重复，0.5g土） DNA提取（DNA SPI试剂盒） 纯 化 （ 用 FAST DNA PREP SYSTEM清除蛋白质和其他杂质 ）获得DNA模板 顺向引物 27F(5’- [6FAM]AGAGTTTGATCCTGGCTCAG- 3’) 和 反 向 引 物 338R(5’- GCTGCCTCCCGTA GGAGT-3’)合成 0.06% 牛血 清白蛋 白， 1.5mM MgCl2 ，2单 位Taq DNA聚合酶，1×PCR缓冲液 ATP, TTP, GTP, CTP 各0.2 mM 10 ng 0.2 uM 94℃初始变性3分钟 94℃变性45秒 55℃退火45秒 72℃延长2分钟 25次 72℃保持7分钟 取1ngPCR产物 宽区聚丙烯酰氨凝胶电泳 PCR-DGGE基本步骤 聚丙烯酰氨变性梯度凝胶电泳 条带分析 建立系统树
DGGE Denaturing Gradient Gel Electrophoresis Separated DNA of same size based on se quence differences Different sequences behave differently at different amounts of denaturing chemical (or heat; see TGGE At some point dNa strand completely sep arate Complete separation is hindered by Gc-cl amp added to one of the PCr primers
DGGE ▪ Denaturing Gradient Gel Electrophoresis ▪ Separated DNA of same size based on se quence differences. ▪ Different sequences “behave differently at different amounts of denaturing chemical (or heat; see TGGE) ▪ At some point DNA strand completely sep arate. ▪ Complete separation is hindered by GC-cl amp added to one of the PCR primers
Gradient Perpendicular to Electrop noesis to Optimize Run Denaturant 少100% LL■■ mm Wild Type Partially melted JIIIII Mutant mutant ITI■ T Partially melted JL willd type Single strands TMIII Double strand
Gradient Perpendicular to Electrop horesis to Optimize Run
DGGE Denaturing Gradient Gel Electrophoresis (DGGE Urea/Formamide gradient Bands were excised Separates double stranded dna based on sequence Eluted DNa was reamplified using PCr Muyzer et al. 1993 Band purity was verified with L DGGE Sequencing and phylogenetic analySIS DGGE Banding patterns are representative of microbial community structure
DGGE •Denaturing Gradient Gel Electrophoresis (DGGE) •Urea/Formamide Gradient •Separates double stranded DNA based on sequence Muyzer et al., 1993 DGGE Banding patterns are representative of microbial community structure •Bands were excised •Eluted DNA was reamplified using PCR •Band purity was verified with DGGE •Sequencing and phylogenetic analysis