DNA实验技术

This method is derived from a procedure developed by Toby Bradshaw and the Poplar Molecular Genetics Cooperative. We have tested the procedure with a variety of Populus species, as well as tobacco and Arabidopsis . The resulting DNA is of sufficiently high quality for PCR (including RAPD), restriction digests, and ligation reactions. However, it is extemely important to use the youngest leaves available for Populus , as DNA from older leaves is often contaminated with compounds that can interfer

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当双链DNA 分子的一条链上产生切口时,E.coli DNA 聚合酶Ⅰ就可将核苷酸连接到切口的3'羟基末端。同时该酶具有从5'→3'的核酸外切酶活性,能从切口的5'端除去核苷酸。由于在切去核苷酸的同时又在切口的3'端补上核苷酸,从而使切口沿着DNA 链移动,用放射性核苷酸代替原先无放射性的核苷酸，将放射性同位素掺入到合成新链中。最合适的切口平移片段一般为50-500个核苷酸。切口平移反应受几种因素的影响: (a) 产物的比活性取决于dNTP ...

Materials: Silica Suspension: add 2 g of silica to 15 ml of H2O wash 3x by centifugation at 2000 x g for 2 min estimate vol of silica and resuspend in 2 vol H2O Silica Wash Solution: 50 mM NaCl, 10 mM Tris 7.5, 2.5 mM EDTA, 50% Ethanol 6 M NaI

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The following protocol is based on our modifications of R. Kraft, J. Tardiff, K. S. Krauter, and L. A. Leinwand. Biotechn . 6(6):544-545, 1988. Inoculate 2-5 ml of L broth containing the appropriate antibiotic from a single bacterial colony. Incubate at 37℃ overnight with vigorous shaking. Follow the Plasmid Quick Prep protocol through the potassium acetate step to isolate plasmid DNA. Add DNAse-free RNAse to 50µg/ml, incubate, 37℃ from 10-30 minutes. Phenol extract the solution, saving the uppe

一、DNA 提取　 SDS 高盐法(方案1) 具体步骤: 称取1 g 土壤，放入研钵中，倒入适量的液氮，立即研磨；再倒入适量的液氮，研磨，重复3 次，使土壤颗粒研成粉末； 将13.5 ml 提取缓冲液(0.1 mol/L磷酸盐 ，0.1 mol/L EDTA ，0.1 mol/L Tris-HCl ，1.5 mol/L NaCl ，1.0 % CTAB) 和50&mu ...

TA Cloning I. Initial mixture 5 μl dd H2O 1 μl PCR product 1 μl ligation buffer 2 μl TA vector (add second to last) 1 μl ligase (add last) Incubate overnight at 15℃. II. Electroporation 1. Chill cuvettes on ice before starting. 2. Set out electrocompetent JS5 cells to thaw on ice. It is important to keep them on ice. 3. Dry one 100 mg/ml AMP plate for each sample. Add 40 μl of xgal and 40 μl of IPTG to each plate. 4. Add 500 μl of SOC or SOB media to each 5 ml snap cap tube. 5. Heat ligations at

Materials: 1.TENS solution: 10 mM Tris (pH to 7.5) 1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve) 0.1 N sodium hydroxide 0.5 % sodium dodecyl sulfate 2.3 M Sodium acetate pH 5.2 3. ...

体外定点突变技术是研究蛋白质结构和功能之间的复杂关系的有力工具，也是我们在实验室中改造/优化基因常用的手段。蛋白质的结构决定其功能，二者之间的关系是蛋白质组 研究的重点之一。对某个已知基因的特定碱基进行定点改变、缺失或者插入，可以改变对应的氨基酸序列和蛋白质结构，对突变基因的表达产物进行研究有助于我们了解蛋白质结构和功能的关系，探讨蛋白质的结构/结构域。而利用定点突变技术改造基因，相信大家也非常熟 ...

“多莉”的诞生，意味着人类可以利用动物的一个组织细胞 ，像翻录磁带或复印文件一样，大量生产出相同的生命体，这无疑是基因工程研究领域的一大突破。 人们剪下植物枝条，扦插到土里，不久就会发芽，长出新的植株，这些植株是遗传物质组成完全相同的植株，这就是“克隆 ”。还有将马铃薯等植物的块茎切成许多小块进行繁殖，由此而长出的后代也是“克隆 &rd ...

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1.grow plants in trays of 96 and leave two spots open (for the PCR controls) 2.harvest 1 to 2 young and green leaves (1cm2 /plant at rosette stage if possible). Use 96 well plates (1 or 2 ml E&K ...

在分子生物学研究中，DNA 的序列分析是进一步研究和改造目的基因的基础。目前用于测序的技术主要有Sanger等（1977）发明的双脱氧链末端终止法和Maxam和 Gilbert（1977）发明的化学降解法。这二种方法在原理上差异很大，但都是根据核苷酸在某一固定的点开始，随机在某一个特定的碱基处终止，产生A，T，C，G四组不同长度的一系列核苷酸，然后在尿素变性的PAGE胶上电泳进行检测，从而获得DN ...

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CTAB TECHNIQUE / Method / Schedule / Protocol (JPB) FOR DNA ISOLATION / DNA EXTRACTION FROM PLANT LEAF / LEAVES SAMPLES (see also DNA RNA double isolation procedure if both DNA and RNA are needed) Rea ...

一、核酸的纯化 在分子克隆 的所有操作中，最基本的操作是核酸的纯化。其关键步骤是去蛋白质，通常只要用酚/氯仿。氯仿抽提核酸的溶液即可。每当需要把克隆 有某一些所用的酶灭活或去除以便进行下一步时，可进行这种抽提。然而，如要从细胞裂解液等复杂的分子混合物中纯化核酸，则要先用某些蛋白水解酶消化大部分蛋白质后，再用有机溶剂抽提。这些广谱的蛋白酶包括链霉蛋白酶及蛋白酶K等，它们对多种天然蛋白均有活性，（1） ...

competent cells (DH5alpha at competency of 108 cfu/µl of pUC18). The transformation procedure it self is very similar to a standard CaCl2 transformation. Place 100 µl of TB buffer in a tub ...

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