DNA实验技术

小于100bp的片断通常在电泳时就比较难观察分辨，需要用分辨率很高的琼脂糖或者丙烯酰胺凝胶。比如Cambrex的NuSieve 3:1或者GTG，或者是大名鼎鼎的MetaPhor琼脂糖。所以实际上对于小片断，可以分为两种情况：一个是真的要回收电泳中特别小的片断，这种情况我们前面有提到，比如Qiagen MinElute系列可以回收70bp以上的片断；而QIAEX纯化介质可以回收40bp以上的小片断，可以根据情况选择。 ...

（一）第一向等电聚焦 1. 从冰箱中取-20℃冷冻保存的水化上样缓冲液（I）（不含DTT，不含Bio-Lyte）一小管（1ml/管），置室温溶解。 2. 在小管中加入0.01g DTT， Bio-Lyte 4-6、5-7各2.5ml，充分混匀。 3. 从小管中取出400ml水化上样缓冲液，加入100ml样品，充分混匀。 4. 从冰箱中取-20℃冷冻保存的IPG预制胶条（17cm pH 4-7），室温中放置10分钟。 5. 沿着聚焦盘或水化盘中槽的边缘至左而右线性加入样品。在槽两端各1cm左右不要加样，中间的样品液一定要连贯。注意：不要产生气泡。否则影响到胶条中蛋白质的分布。 6. 当所有的蛋白质样品都已经加入到聚焦盘或水化盘中后，用镊子轻轻的去除预制IPG胶条上的保护层。 7. 分清胶条的正负极，轻轻地将IPG胶条胶面朝下置于聚焦盘或水化盘中样品溶液上，使得胶条的正极（标有+）对应于聚焦盘的正极。确保胶条与电极紧密接触。不要使样品溶液弄到胶条背面的塑料支撑膜上，因为这些溶液不会被胶条吸收。同样还要注意不使胶条下面的溶液产生气泡。如果已经产生气泡，用镊子轻轻地提起胶条的一端，上下移动胶

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 ...

This protocol is intended to make shotgun libraries from BACS that are to be fully sequenced and assembled. To minimize chimeric clones an adaptor method is used. There are simpler protocols if just r ...

FINGERPRINTING PROTOCOL (AGAROSE GEL) * Restriction digests consist of: 15.75 ml ddH2 O 1 µl 10 X buffer B (Boehringer Mannheim) 0.25 µl HindIII (40 U/µl) 3 µl DNA Set up ...

第一节 概 述 一. DNA的限制性内切酶酶切分析 限制性内切酶能特异地结合于一段被称为限制性酶识别序列的DNA序列之内或其附近的特异位点上，并切割双链DNA。它可分为三类:Ⅰ类和Ⅲ类酶在同一蛋白质分子中兼有切割和修饰(甲基化)作用且依赖于ATP的存在。Ⅰ类酶结合于识别位点并随机的切割识别位点不远处的DNA，而Ⅲ类酶在识别位点上切割DNA分子，然后从底物上解离。Ⅱ类由两种酶组成: 一种为限制性 ...

DNA absorbs ultraviolet light due to its highly conjugated nature. DNA may thus be easily quantitated in a UV spectrometer. Typically 1 OD260 (i.e. a solution having an absorbance of one unit at 260 n ...

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本方法的核心部分是医科院基础所的生化脂蛋白组的吴刚老师所创，我在其基础上进行了一些改动，主要是DNA回收上采取了更简单的方法。（本方法最适用于双酶切制作片断并进行克隆 的情况。对于分步酶切制作片断，也可以使用本方法，但需要加倍起始酶切DNA的量。） 一、片断平移的克隆 （也适用于多片断连接） 简介：将用作载体的质粒A（制作大片断）酶切3μL ，要切出小片断的质粒B酶切7μL；琼脂糖 ...

I have been using an alternative to wedge gels that saves acrylamide, cuts gel drying time to 20 min and gives as good or better band squashing at the bottom of the gel. It was published in BioTechniques about 3 or 4 years ago (email me if you want the reference). The method goes as such: Run the gel with 0.5X TBE in the top tank and normal 1 X TBE in the bottom tank. Just after the last (or in the case of one load - the only) load has run into the gel, put a half volume of 3 M sodium acetate in

16 µl BigDye Terminator Ready Reaction Mix 1 µg BAC DNA (determined from gel) 10 pmol primer water to 40 µl Heat tubes at 95℃ for 5 min., then perform 30 cycles of: 95℃ x 30 sec 55℃ x 10 sec 60℃ x 4 min Run BACs on a 377 and load the entire sample after clean up. If you use a 3700, the loading would be very different. This IS the double recipe. Normally a reaction is only 20 µl. For sequencing we use ABIs "BigDye Terminator Cycle Sequencing Ready Reaction Kits v2.0 with AmpliTaq DNA Polymerase",

macerate tissue in Eppendorf tube without butter at RT add 400 m l extraction buffer vortex for 4 sec leave sample at RT until other samples are ready (> 1 h) spin in microfuge for 1 min transfer 300 m l of supernatant to different Eppendorf tube (prefilled with 300 m l isopropanole) mix and leave at RT for 2 min spin for 5 min vacuum dry pellet and take up in 100 m l TE use 1-2.5 m l for PCR Remarks: DNA is stable for one year at 4℃ Solutions: Extraction buffer: 200 mM Tris-HCl pH 7

The principle and procedures of RLGS method was first described by Hatada et al. (1991) and its improvement was described by Asakawa (1996). Basically based on their procedures ...

酵母 菌基因组 DNA的提取 一：仪器： 同方法一 二：试剂： SE缓冲液（1M山梨醇，0.1MEDTA pH7.5）；溶菌酶（50mg/ml）；20％PVP；蛋白酶K缓冲液（10mM Tris pH7.6 0.5% SDS 1mM EDTA）；其余同前 三：操作 1.5ml 对数生长期细菌细胞 离心，12000rpm，1-2min 沉淀 溶于590ulSE缓冲液中混匀+1 ...

PCR产物克隆大致分为两类，即平头连接和粘头连接。 平头连接是将制备好的平头载体和补平或削平的PCR 产物直接进行连接。载体可用EcoR V或Sma I切成平头；PCR 产物纯化后，可以在22℃用DNA聚合酶I作用30min（利用该酶所具有的3’→5’外切酶活性和5’→3’的聚合酶活性）。如果要求不高，PCR 产物也可不加处理。如 ...

Protocol for Annealing OligonucleotidesOligo Name:Lot Number:Total nmol:Volume of Annealing Buffer added:Oligo Name:Lot Number:Total nmol:Volume of Annealing Buffer added:Annealing Buffer: 10mM Tris, pH 7.5 - 8.0, 50mM NaCl, 1mM EDTA1xTE Buffer: 10mM Tris, pH 7.5 - 8.0,1mM EDTA.1.R ...

An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average every DNA molecule is c ...

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Bst DNA polymerase-catalyzed radiolabeled two-step sequencing reactions (26) are modified from those presented earlier (25) by altering the absolute amounts and the relative deoxy/dideoxynucleotide ra ...

PEG Preparation of Plasmid Plasmid isolated by this procedure can be used routinely for electrophoretic analysis, restriction endonuclease digestion and transformation of E. Coli., sequencing, PCR and most other molecular biological techniques. The procedure is a modification of the rapid alkaline lysis method of Ish-Horowitz and Burke (1981). You will need 3 basic solutions:- Solution I: 50mM glucose, 25mM Tris.Cl, pH 8, 10mM EDTA Solution II: 0.2M NaOH, 1%(w/v) SDS Solution III: 3M potassi