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[求助]
PCR检测菌株的耐药性
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如何用PCR对菌株的耐药性进行检测？在检测前怎样提取细菌的DNA?我目前正在做药敏试验，来寻找具有耐药性的菌株。然后再用PCR进行进一步的验证。那么，怎样提取菌株的DNA?怎样进行PCR?谢谢各位！ 发自小木虫Android客户端 |
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According your description above, the drug-resistant bacteria will be identified by 16S rDNA sequence analysis. The DNA of resulting bacteria will be extracted with 3% CTAB extraction protocol. The amplication of 16rDNA sequences will be made with general primers P0 (5'-GAGAGTTTGATCCTGGCTCAG-3') and P6 (5'CTACGGCTACCTTCTTACGA-3'). PCR amplification will be performed in a 25ul reaction mixture containing 100ng genomic DNA, 3uM of each primer, 1X PCR buffer (supplied with Taq polymerase), 25mM MgCl2, 2mM dNTPs and 0.25U of Taq polymerase (TaKaRa Biotech, Dalian, China). The thermal cycling program will be run on an Eppendorf Mastercycler using the following conditions: 5 min initial denaturation at 94 degrees Celsiu, followed by 30 cycles of 30s denaturation at 95 degrees Celsiu, 30s annealing at 55 degrees Celsiu, 90s extension at 72 degrees Celsiu and a final 7min extension at 72 degrees Celsiu. A negative control using sterilized water as template will be included in the amplifications. The PCR products will be separated in 1% agarose gels by electrophoresis, purified, cloned and finally sequenced by Sangon Biotech. Sequence similarity will be searched against the GenBank database using the National Center for Biotechnology Information BLASTN Network Server. |
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2016-01-08 16:41:44, 50.16 K
2楼2016-01-08 16:41:50
