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第一节 概 述 DNA分子水平上的多态性检测技术是进行基因组研究的基础。RFLP(Restriction Fragment Length Polymorphism限制片段长度多态性)已被广泛用于基因组遗传图谱构建、基因定位以及生物进化和分类的研究。RFLP是根据不同品种（个体）基因组的限制性内切酶的酶切位点碱基发生突变，或酶切位点之间发生了碱基的插入、缺失，导致酶切片段大小发生了变化，这种变化可以 ...

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 ...

DNA 是遗传信息的载体，是最重要的生物信息分子，是分子生物学研究的主要对象，因此DNA 的提取也应是分子生物学实验技术中的最重要、最基本的操作，如不能有效的完成DNA 提取方面的工作，那就根本谈不上进行分子生物学方面的实验。在DNA 提取过程中应做到1，根据不同研究需要，保证结构的相应完整性；2，尽量排除其它大分子成分的污染(蛋白质、多糖及RNA 等)；3，保证提取样品中不含对酶有抑制作用的有机 ...

无论是从一个胚胎细胞分化为不同的组织器官，或者是从正常的组织突变为肿瘤组织，都可能涉及在同一基因组 背景下不同基因的差异性表达。寻找差异表达的基因就有可能揭示细胞分化的机制或者肿瘤的成因，因而也就成为一项热门的技术。 寻找差异表达的基因有不少方法，抑制消减杂交（SSH）是比较有效的一种方法。以Clontech的PCR-Select cDNA Subtraction Kit为例：将样品（teste ...

一、 建立一个标准的酶切反应目前大多数研究者遵循一条规则，即10个单位的内切酶可以切割1μg不同来源和纯度的DNA。通常，一个50μl的反应体系中，1μl的酶在1X NEBuffer终浓度及相应温度条件下反应1小时即可降解1μg已纯化好的DNA。如果加入更多的酶，则可相应缩短反应时间；如果减少酶的用量，对许多酶来说，相应延长反应时间（不超过16小时）也可完全反应。二、 选择正确的酶不言而喻，选择的酶在底物DNA上必须至少有 ...

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） ...

Marian Price-Carter, 9/7/00Day 1 : Start overnight cultures in assay medium.Negative control : cells lacking b -galactosidase, such as LT2; positive control : cells with high enzyme activity.Day 2 : Dilute cells 1/100 in fresh medium, grow to mid-log.1 Prepare solutions: Z buffer, phosphate buf ...

Materials: phenol:chloroform (1:1) chloroform Add an equal volume of buffer-saturated phenol:chloroform (1:1) to the DNA solution. Mix well. Most DNA solutions can be vortexed for 10 sec except for high molecular weight DNA which should be gently rocked. (If using Phase-Lock Gel, follow procedure M.1 ) Spin in a microfuge for 3 min. Carefully remove the aqueous layer to a new tube, being careful to avoid the interface. (Steps 1-4 can be repeated until an interface is no longer visible) To

Footprinting Footprinting is a method for determining the exact DNA sequence to which a particular DNA-binding protein binds. Examples: hormone-receptor complexes that bind to their hormone response elements transcription factors that bind eukaryotic operators, enhancers, and silencers the lac repressor that shuts down the lac operon in E. coli [Discussion of the lac operon] How, for example, does one determine the DNA sequence to which the lac repressor binds? The procedure: Clon

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Wash one 0.45um Millipore nitrocellulose filter (Cat# HAWP01300) in 1ml 0.4M KOH for 1 min; wash in 3ml dH2 O for >1min. Assemble filter apparatus: white plastic bottom support/funnel nitrocellulos ...

限制酶在各种NEBuffer 中的活性 (NEBuffer Activity Chart for Restriction Enzymes) 对应每一种限制性内切酶，New England Biolabs 均提供彩色盖子的10X NEBuffer，以保证100%活性。下面表格中列出每一种酶及其随酶提供的最适宜的NEBuffer。同时也列出每种酶在四种不同的NE ...

DNaseI Footprintint Solutions 10X Binding Buffer 200 mM Tris 8.0 200 m l 1M Tris pH 8.0 500 mM NaCl 100 m l 5M NaCl 10 mM EDTA 20 m l 0.5 M EDTA pH 8.0 680 m l Q store at room temperature DNaseI Dilut ...

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TE solution 10 mM Tris (pH to 7.5) 1 mM EDTA (pH to 8.0 to dissolve) 2.Frozen agarose gel piece containing the desired DNA fragment Supplies: 1.Micropipetter and tips 2.Microcentrifuge and tubes 3.Spatula 4.PCR machine and the 600-ul tubes for use in the machine 5.Ultrafree-MC(R) filter units, 0.45 μm Procedures: 1.Quick thaw the gel piece in a 600-μl tube using the PCR machine at 37 ℃ 2.Macerate the gel piece with a spatula. 3.Transfer into the sample cup of the filter unit. 4.Spin th

Sephadex G-50 spun column purification Spin column purification can be used to change buffers without a concomitant change in solution volume to remove protein contaminants or to purify plasmid DNA s ...

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Pouring the Gel Outline: Pouring this big & thin 6% acrylamide gel ("Mother of all gels") is quite a challenge and probably the reason why smart whimps by them ready to use. Supplies & ...

This protocol works well from a 5 ml starting culture or a 1 ml starting culture (adjust volume accordingly). Solutions 2X YT Media 16 g tryptone 10 g yeast extract 5 g NaCl 1 ml 1N NaOH up to 1 liter with Q Ampicillin Stock (1000X) 0.15 g ampicillin 1 ml Q can be stored at 4℃ for several weeks Tetracycline Stock (1000X) 15 mg tetracycline 500 m l EtOH 500 m l Q vortex to dissolve and store at 4℃ Kanamycin Stock (1000X) 50 mg kanamycin 1 ml Q can be stored at 4℃ for several weeks 20% PEG 8000/ 2

1. Add 400 μl of TE buffer then 400 μl of 1-butanol to the oligonucleotide glass vial. 2. Vortex well, then spin down in tabletop centrifuge for 10 minutes at ~2,000. rpm¹s. 3. Remove top, butanol layer with a sterile pipette tip and discard. 4. Add another 400 μl of 1-butanol. 5. Vortex well, then spin down as above in tabletop centrifuge. 6. Remove top, butanol layer and discard. 7. Transfer aqueous phase into a new 1.5 ml tube. 8. Dry in a speed vac for about 5 minutes to remove all of the b