Extraction of DNA from Fermented Wi

Extraction of DNA from Fermented Wine Samples and Identification of Lactobacillus plantarum and Oenococcus oeni by PCR-DGGE. At each step of wine fermentation, duplicate 10-g samples were homogenized in a stomacher bag with 10 mL of salinepeptone water for 1 min. After each preparation had settled for 1 min,two 1.8-ml subsamples were placed in 2-mL screw-cap tubes and total DNA was extracted using a Powersoil DNA extraction kit (Cabru,Milan, Italy) according to the manufacturerÕs procedure. After DNA was isolated, 500 lL of phenol–chloroform–isoamyl alcohol (25:24:1; pH 6.7; Sigma) was added to each tube and the tubes were then centrifuged at 12,000 g at 4°C for 10 min, the aqueous phases was collected, and DNA was precipitated with 2.5 (vol/vol) ice-cold absolute ethanol and 1/10 (v/v) NaAcetate 3M pH 3.5. The DNA was collected by centrifugation at 14,000 g at 4°C for 10 min, and the pellet was dried under vacuum at room temperature. Fifty microliters of sterile water was added and the preparation was incubated for 30 min at 45°C to facilitate nucleic acid solubilization. One microliter of DNase-free RNase (Invitrogen) was added to digest RNA, during incubation at 37°C for 1 h. PCR conditions employed were as reported above. In order to test the absence of Taq polymerase inhibitors, primers pA and pH were used to amplify a region of approximately
1 kb of eubacterial 16S-rDNA [10].

結果 (中文) 1:

发酵酒样品和乳酸杆菌鉴定的植物乳杆菌和由 PCR-DGGE 酒酒球菌 DNA 提取。酒发酵的每一步，重复 10 g 样品均在 10 毫升的 salinepeptone 水 1 分钟抹胸袋质化后。每个准备定居 1 分钟后，两个 1.8 毫升标本被放置在 2 毫升螺旋帽管和总 DNA 提取使用 Powersoil DNA 提取试剂盒 （Cabru，意大利米兰） 按照制造商的程序。DNA 是孤立的后, 500 lL 苯酚-氯仿-异戊醇 （25:24:1; pH 6.7;西格玛） 被添加到每个管和管分别在 12,000 g 在 4 ° C，10 分钟后离心，收集了水相中，然后 DNA 沉淀 2.5 （vol/卷） 冰冷无水乙醇和 1/10 (v/v) NaAcetate 3 M ph 值 3.5。DNA 由在 14,000 g 在 4 ° C，10 分钟，离心收集和颗粒真空在室温下干燥脱水。五十办到的无菌水被添加和准备孵化 30 分钟在 45 ° C，以促进核酸溶。一微升的无头的核糖核酸酶 (Invitrogen) 被添加到摘要 RNA，37 ° C 1 h.PCR 条件受雇在孵化过程中，如上文所述。为测试 Taq 聚合酶抑制剂，引物没有 pA 和 ph 值对进行大约放大的区域1 kb 的 eubacterial 16S rDNA [10]。

結果 (中文) 3:

DNA样品和植物乳杆菌发酵酒和酒类酒球菌PCR-DGGE提取鉴定。在葡萄酒发酵的每一步，重复10克样品匀浆在抹胸袋10毫升水1分钟后salinepeptone各制备了1分钟，两1.8-ml样本被放置在2毫升的螺丝帽管和总DNA，利用总DNA提取试剂盒提取（cabru，米兰，意大利）根据制造商Õ程序。在DNA中分离，500会––苯酚氯仿异戊醇（提取；pH为6.7；σ）被添加到每个管和管，然后离心12000克在4°C 10分钟，水相进行收集，和DNA沉淀用2.5（体积/体积）冰冷的绝对乙醇和1 / 10（v/v）naacetate 3M pH 3.5。收集4克10°C离心14000°C，并在真空下干燥的粒料。五十微升的无菌水的添加和制备孵育30分钟45°C促进核酸增溶。1微升无DNA酶核糖核酸酶（Invitrogen）被添加到消化RNA，在孵化37°C 1小时PCR条件为上述报道。为了测试Taq聚合酶抑制剂的情况下，引物PA和pH值是用来放大约一区真细菌16S rDNA基因的[ 10 ] 1 KB。