基于16S rDNA的系统发育分析.ppt

16S rDNA: a suitable phylogenetic marker 16S rDNAs 存在于所有的生物中 16S rDNAs 具同源性而且功能类似 16S rRNA 基因在遗传上相对稳定 16S rDNAs 分子大小适当 核蛋白体是肽链合成的场所 Protocol for PCR Amplification of 16S rDNA Preparation of template DNA Primer PCR mixture (50μl volume) Thermal cycle parameters in PCR program Purification and Sequencing of PCR amplified 16S rDNA Preparation of DNA template Standard method Boiling treatment Boiling treatment 1. After grown on liquid medium, 1ml culture was centrifuged with 12,000r/min for 1min and resuspended in 100μl ddH2O. The supernatant was used directly as template for PCR amplification after boiled for 1min and centrifuged with 12,000r/min for 5min. 2. Or 1ml culture was used directly as template for PCR amplification after boiled for 1min. 3. Or after grown on solid medium, one loop of cells from a single bacterial colony suspended in 100μl ddH2O. The supernatant was used directly as template for PCR amplification after boiled for 1min and centrifuged with 12,000r/min for 5min. Primer The rRNA www server http://rrna.uia.ac.be/ the forward primer BSF8/20 (primering site 8-27) 5'-AGAGT TTGAT CCTGG CTCAG-3' the reverse primer BSR1541/20 (primering site 1541-1522) 5'-AAGGA GGTGA TCCAG CCGCA-3' Centrifuged with 4℃, 12,000r/min for 5min Diluted in ddH2O to about 100 μg/ml (1 OD primer + 300μ l ddH2O) PRIME primer5.0用于本地引物设计 PCR mixture ( 50μl) Thermal cycle parameters in PCR program 1.??94℃ 2min 2.? 94℃ 1min, 56℃ 1min, 72℃ 2min 3.? repeat the second step for 29 cycles 4.? 72℃ 10min 5.??4℃ 10min 6.??stored in room temperature Key steps in PCR 1. Boiling treatment 不形成芽孢的细菌 1-2min 形成芽孢的细菌 5min 形成芽孢的嗜热细菌 10-15min 2.?ddH2O 3. Template 4. Taq polymerase 5. CK: Negative control and Positive control Purification and Sequencing of PCR amplified 16S rDNA Primer for sequencing Forward primer Reverse prime Free software and databases for phylog