DNA提取与纯化

ECK Description This method was suggested in a BRL "Focus" article several years ago (Zeugin & Hartley, 1985). It is a useful way to avoid coprecipitation of proteins and accumulation of salt in the final dried preparation. Protocol 1. Crude preparations: Add 0.5 volumes of 7.5 M NH4OAc. Pure preparations: Add 0.1 volumes of the same. 2. Add an amount of 95% ethanol equal to 2.5 times the new volume. 3. Continue per your favorite protocol. Note that a subsequent BRL article (-, 1982) pointed

琼脂糖凝胶电泳片断回收的常用方法20世纪70年代是一个奠定现代分子生物学的时代，1973年冷泉港实验室的Joseph Sambrook和Phillip Sharp发明了利用琼脂糖凝胶分离DNA 和EB染料观测DNA 相结合的技术。现在，琼脂糖和聚丙烯酰胺凝胶电泳是分离、鉴定和纯化核酸和蛋白质片断的标准方法。这里首先介绍的是琼脂糖凝胶电泳片断回收的常用方法：1.柱回收试剂盒：可谓目前最简单快速的回收方法，只需要将电泳凝胶中 ...

Recovery of DNA from Low Melting Point Agarose Gels 1.Run digestion products on 0.7% LMP agarose gel in 1X TBE (it's nice to have at least 1ug of the fragment you want). LMP agarose is fragile; pour gel with EtBr 2.Let solidify in cold room. Be sure to overlay the gel with buffer before pulling out the comb, to prevent damage to the wells 3.Run gel in cold room, 100V 4.Cut out fragment of interest with clean razor blade and remove all excess agarose from the DNA. 5.Use long wave UV to visualize

'Phenol' as used is Analar grade. Phenol should be melted at 65℃,8-hydroxyquinoline added to a final concentration of 0.1%, and equilibrated three times with an equal volume of 1M Tris.HCl, pH 7.0. The final Tris wash is replaced with TE (10mM Tris, 1mM EDTA, pH8.0) and the phenol stored in the dark at 4℃. 8-hydroxyquinoline is added to prevent oxidation of the phenol and to act as an inhibitor of RNases. 'Chloroform' as used is a water saturated 24:1(v/v) mixture of chloroform and isoamyl alco

一、导论 质粒提取的原理:转自复旦大学一位老师的帖子，后面是方法介绍 碱裂解法从大肠杆菌制备质粒，是从事分子生物学研究的实验室每天都要用的常规技术。可是我收研究生十几年了，几乎毫无例外的是我那些给人感觉什么都知道的优秀学生却对碱法质粒抽提的原理知之甚少。追其原因，我想大概是因为《分子克隆》里面只讲实验操作步骤，而没有对原理进行详细的论述。这是导致我的学生误入歧途的主要原因。后来我发现其实是整个中 ...

Salmon Sperm DNA1.Dissolve 1 g salmon sperm DNA in 100 ml H2 O. 2.Autoclave (20 minutes) and aliquot in 1 ml/tube 3.Store at -20℃. (common freezer for stock solutions)

从琼脂糖凝胶中回收DNA ，是一种简单不过的常规实验操作。但是由于胶回收的质量和数量直接影响后继的一系列实验――比如酶切连接、转化筛选、测序或者PCR扩增、标记乃至显微注射等等，所以这一步的操作也显得非常重要。 胶回收的关键参数 胶回收的质量直接影响后继实验的成功与否。要想做好胶回收，无论是自己亲力亲为还是借助目前五花八门的胶回收试剂，最基本的评定标准无外乎这么几个:质量（ 回收产物的纯度和浓度） ...

（一）质粒DNA 的提取及鉴定 1．收获细菌 （1）将2ml含相应抗生素的LB液体培养基加入到通气良好的15ml的试管中，接入一单菌落，于37℃剧烈振荡培养过夜。 （2）将1.5ml培养物倒入1.5ml离心管中，用台式离心机于4℃以12000g离心5min，将剩余的培养物贮存于4℃。 （3）吸尽培养液，使细菌沉淀尽可能干燥。 2．碱裂解法小量抽提质粒 （1）将细菌沉淀[上述步骤（3）所得]重悬于100μl预冷的溶液Ⅰ（50mmol/L葡萄糖，25mmol/l Tris �HCl pH8.0, 10mmol/L EDTA）中，剧烈振荡。 （2）加200μl新配制的溶液Ⅱ（0.2mol/l NaOH, 1%SDS），缓和振荡，水浴5min。 （3）加150μl预冷溶液Ⅲ（50mol/L KAC 60ml, 冰乙酸11.5ml,水28.5ml），缓和振荡，冰浴5min。 （4）4℃以12000g离心5min，将上清转移到另一离心管中。 （5）可作不可作：加等量饱和酚，氯仿，振荡混匀，重复2，（4）。 （6）用2倍体积无水乙醇室温沉淀双链DNA ，振荡混合，于室温放置2min。 （7）4℃以1

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This protocol describes a method for isolating DNA from plant tissue. Procedure 1. Preheat the CTAB Isolation Buffer at 60°C. 2. Grind 2 g of fresh, leaf tissue to a powder in Liquid Nitrogen in a chilled mortar and pestle. 3. Scrape the powder into a chilled 50 ml tube. 4. Add 3 to 5 ml CTAB Buffer per gram tissue to the tube. 5. Incubate the sample at 60°C for 30 min with occasional gentle swirling. 6. Add the same volume of Phenol:Chloroform to the sample, gently swirling. 7. Centrifuge at 16