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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

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

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

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 ...

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 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 ...

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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",

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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 ...

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

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 ...

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 ...

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测定DNA 的核苷酸序列是分析基因结构与功能关系的前提。从小片段重叠法到加减法、双脱氧链终止法、化学降解法、自动测序，DNA 测序技术发展很快。目前在实验室手工测序常用Sanger双脱氧链终止法。Sanger法就是使用DNA 聚合酶和双脱氧链终止物测定DNA 核苷酸序列的方法。它要求使用一种单链的DNA 模板或经变性的双链DNA 模板和一种恰当的DNA 合成引物。其基本原理是DNA 聚合酶利用单链的DNA 模板，合成出准确互补链，在合成时，某种dNTP换成了ddNTP，这时，DNA 聚合酶利用2’，3’-双脱氧核苷三磷酸作底物，使之掺入到寡核苷酸链的3’末端，导致3’末端无3＇-OH，从而终止DNA 链的生长，双脱氧核苷酸的种类不同，掺入的位置不同就造成了在不同的专一位置终止的长度不同的互补链。通过掺入放射性核苷酸和聚丙烯酰胺凝胶电泳，即可读出模板DNA 的互补链序列。 一、 试剂准备 1.硅化液：四氯化碳250ml，二氯二甲基硅烷25ml。 2.6%变性PAGE胶的配制：丙烯酰胺 28.5g，N，N’-亚甲基双丙烯酰胺1.5g，10×TBE 50ml，尿素210g，加ddH2O至50

在自然条件下,很多质粒都可通过细菌接合作用转移到新的宿主内,但在人工构建的质粒载体中,一般缺乏此种转移所必需的mob基因,因此不能自行完成从一个细胞到另一个细胞的接合转移。如需将质粒载体转移进受体细菌，需诱导受体细菌产生一种短暂的感受态以摄取外源DNA。

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 ...

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 ...

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