A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single...A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Taq polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC234 from cucumber （Cucumis sativus L.）.展开更多
Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher ampli...Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 3 hr at 30°C or 1 hr at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentration, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not primarily due to the reduced polymerase concentration resulted from the above interaction. This study provided another good start to investigate potential implications of quantum dots in key molecular biology techniques.展开更多
为了优化以土壤微生物DNA为模板的PCR反应条件,采用E.Z.N.A.soil DNA kit试剂盒提取土壤微生物总DNA,对16SrDNA V3可变区的PCR反应体系和反应条件进行优化。主要从DNA模板用量、引物浓度、退火温度、热启动方式4个方面进行筛选试验,最...为了优化以土壤微生物DNA为模板的PCR反应条件,采用E.Z.N.A.soil DNA kit试剂盒提取土壤微生物总DNA,对16SrDNA V3可变区的PCR反应体系和反应条件进行优化。主要从DNA模板用量、引物浓度、退火温度、热启动方式4个方面进行筛选试验,最后得出最适宜的土壤DNA扩增体系为:10.5ng模板DNA、5μL 10×buffer、4μL 2.5mmol/L dNTP、10μmol/L引物各1.5μL,2.5UTaq酶,加无菌ddH2O补足至50μL;PCR循环程序为:94℃预变性5min;94℃变性1min,52℃退火1min,72℃延伸70s,29个循环;72℃延伸5min。试验结果表明:选择热启动方式和合适的退火温度是获得高质量PCR产物的关键。展开更多
基金partially supported by the Program 30470120,30671419,30700541 from the National Natural Science Foundation of Chinaby the 863 Programs 2006AA10Z108,2006AA100108 from the Ministry of Science and Technology of China+2 种基金by the Ph.D Funding 20050307009 from the Ministry of Education of Chinaby the Program BK2006139 from the Natural Science Foundation of Jiangsu Provinceby Research Fund KJ05006 from Nanjing Agricultural University,China.
文摘A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Taq polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC234 from cucumber （Cucumis sativus L.）.
文摘Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 3 hr at 30°C or 1 hr at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentration, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not primarily due to the reduced polymerase concentration resulted from the above interaction. This study provided another good start to investigate potential implications of quantum dots in key molecular biology techniques.
