DNA实验技术

For every 4 mls of culture, dissolve the BAC DNA pellet in 40 µl of water. for example: Usually each BAC is grown in 20 mls LB/CM total, then is dispensed into one Autogen tube (4 mls in each of the 5 tubes). After miniprep, add 40 µl of water to each tube (200 µl total for each BAC). Vortex the Autogen tube and let sit for at least 0.5 hour. Then pool the 5 samples into one for each BAC. check the BAC DNA for quality and quantity by digesting 5 µl of the DNA in a 20 µl reaction: 5.0 µl DNA 2.0

This is a rapid method for chemical DNA sequencing which is commonly used as ladder for footprinting reactions or for sequencing of short DNA oligonucleotides. Reference: Bencini et al. (1984) Biotechniques 2: 4-5. Steve Hahn/Hahn Lab The method below works well for Sequencing of DNA of greater than ~40 bp. Typically, about 150 bases of sequence can be read from analysis on a 6-8% urea acrylamide gel. For sequencing of short oligonucleotides, the reaction times should be increased as noted below

1. Prepare Whatman DE-52 according to manufacturers specifications. Equilibrate at store with 0.02% Sodium azide. 2. Plug a 1 mL (blue) pipet tip with Siliconized glass wool. Add 500 ml of 50:50 DE-52 ...

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 ...

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 ...

Wear gloves throughout and work in radiation area. Monitor area before and after use. Mix the following in an eppendorf tube: 1. 0.5 microgram oligonucleotide dissolved in H2O. 2. 3 microliters 10x kinase buffer. 3. 2 microliters 32 P ATP from ICN (>5000 ci/mmole). 4. H2O so that the final volume is 30 microliters. Add 25 units T4 polynucleotide kinase and incubate 60 min at 37 ℃. Purify labeled Oligonucleotide away from unincorporated ATP Currently, we use mini Quick Spin Oligo Columns (#1 8

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

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'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

A simple reversible way to a stop restriction reaction is by adding EDTA which chelates Mg2+ thereby preventing catalysis. If further manipulations of the digested DNA are to be performed the restric ...

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 ...

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The following guidelines should be taken into account when designing modified oligonucleotides. 1.Sequence Length - SYNTHEGEN can synthesize oligonucleotides from 5 to 110 bases in length. Most seque ...

Pyrosequencing是对短到中等长度的DNA序列样品进行高通量的、精确和重复性好的分析的技术。 第一步 ――测序引物和PCR扩增的、单链的DNA模板杂交，与酶―DNA聚合酶（DNA polymerase）、ATP硫酸化酶（ATP sulfurylase）、荧光素酶（luciferase）、三磷酸腺苷双磷酸酶（apyrase）―和底物―adenosine 5´ phosphosu ...

This technique can be used to isolate overlapping DNA fragments starting with a previously cloned DNA fragment that maps near a gene of interest (dark red). The walk is continued until a clone containing the desired gene is identified. In this example, the chromosomal DNA fragments are cloned in λ pha ...

The pGL3 Luciferase Reporter Vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be cis-acting, such as promoters and enhancers, or trans-acting, such as various DNA-binding factors. The backbone of t ...

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)

UV shadowing is a technique for visualizing nucleic acids separated on acrylamide/urea gels. The technique utilizes shortwave UV light (254 nm) and a fluor-coated TLC plate. We recommend UV shadowing as the method of choice for gel purification of nonisotopic probes synthesized with Ambion's MAXIscript™ and BrightStar™ Psoralen-Biotin Kits. The alternative to UV shadowing is staining with ethidium bromide or acridine orange and requires subsequent extraction of the dye. The detection limit of UV