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QUOTE julianne: been using sod acetate- isoprop precipiation with no problems all the while. QUOTE Turtle: my suggestion would be to ethanol precipitate QUOTE for PCR product purification used for clo ...

I did stupid thing after cloning a sequence into a plasmid just find out not in frame with the following sequence. just need insert one single nucleoacid will be ok. Any method to insert only one nucl ...

Hi can anyone answer this I am having problems with my southern hybridization. I am wondering if the DNA transfers to my membrane. Is it possible that the DNA can wash into the solutions when denaturi ...

Materials: Silica Suspension: add 2 g of silica to 15 ml of H2O wash 3x by centifugation at 2000 x g for 2 min estimate vol of silica and resuspend in 2 vol H2O Silica Wash Solution: 50 mM NaCl 10 mM ...

Caltech Genome Research LaboratoryCalifornia Institute of Technology http://www.tree.caltech.edu/protocols/agfp.html Restriction digests consist of: 15.75 ml ddH2O 1 µl 10 X buffer B (Boehring ...

Caution: EtBr is a powerful mutagen and is moderately toxic. Wear gloves when working. When EtBr conc. is >0.5 mg/ml: 1.Add sufficient water to reduce the concentration of EtBr to <0.5 mg/ml. ...

I am working with very small amount human tissue. Is there anyone who had the experience of using glycogen with 100% ethanol in pheno/chloroform DNA extraction protocol ? Does it realy work to increas ...

Hancock Laboratory Methods. Department of Microbiology and Immunology University of British Columbia British Columbia Canada http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=67 METHOD: 1.Run gel a ...

Sample Preparation Cheek cells are obtained by rinsing the mouth with 25mls of any commercial mouth wash solution available for about 30sec the first thing in the morning. It is important not to brus ...

Hello group! I want to insert a small 30 bp sequence into a vector of mine using ds-oligo. I've created the oligos so that once annealed they have the correct overhangs of the restriction sites. As I' ...

Donis-Keller Lab Manual Department of Genetics in Washington University School of Medicine http://hg.wustl.edu/hdk_lab_manual/plasmid/plsmid08.html Purpose: To utilize competent E. coli bacteria to r ...

I usually use Linear polyacrylamide as a carrier for DNA precipitation by EtOH. I have read about using glycogen as a carrier I would like to know if anyone knows a method avoiding not to use acrylami ...

10X TBE per liter Sequencing dye ------------------- ---------------- &nb ...

一．原理 (1) 以目的mRNA 为模板，使用Oligo dT-3sites Adaptor Primer 进行反转录反应，合成1st Strand cDNA。 (2) 使用含有KpnI、XbaI、BamH I 酶切位点的上游特异性引物和3sites Adaptor Primer进行PCR反应。 (3) 使用限制性酶（KpnI、XbaI或BamH I）对PCR产物进行酶切反应。 (4) 选用适当载 ...

Application of Electroporation in Gene Transfer and Animal Embryo Cloning 郝新保 范清宇 Hao Xin-bao Fan Qing-yu 第四军医大学全军骨肿瘤研究所 西安 710038 Institute of Orthopedic Oncology Fourth Military Medical Universit ...

一、从在含20 mg/l Kanamycin和15 mg/l Gentamycin 6 ml的液体LB培养基中接种Agrobacterium 细胞，然后在28℃、200-220 rpm培养24 h。准备进行质粒提取。 质粒提取前，检查裂解液（Lysis Solution）是否有沉淀，如果有，要在65℃下溶解。对试剂盒中的稀释溶液（Elution Solution）（用Qiagen公司的EB（5 m ...

一、大肠杆菌质粒DNA的提取 质粒DNA的提取是从事基因工程工作中的一项基本实验技术，但提取方法有很多种，以下介绍一种最常用的方法： 碱裂解法：此方法适用于小量质粒DNA的提取，提取的质粒DNA可直接用于酶切、PCR扩增、银染序列分析。方法如下： 1、接1%含质粒的大肠杆菌细胞于2ml LB培养基。 2、37℃振荡培养过夜。 3、取1.5ml菌体于Ep管，以4000rpm离心3min，弃上清液。 ...

1实验原理 DNA重组分子在体外构建完成后，必须导入特定的受体细胞，使之无性繁殖并高效表达外源基因或直接改变其遗传性状，这个导入过程及操作统称为重组DNA分子的转化。对于不同的受体细胞，往往采取不同的转化战略。 1.1 DNA重组分子的常用转化方法 1.1.1 Ca 2 诱导转化 1970年Mandel和Higa发现用CaCl2处理过的大肠杆菌能够吸收l噬菌体DNA，此后不久，Cohen等 ...

质粒是存在于细菌染色体外的一个或多个能独立复制并稳定遗传的小型环状双链DNA分子，其分子量一般在0. 2～10kD 范围内。由于质粒分子小，便于分离和提取，可以携带目的基因进入细菌、动物细胞或植物体内进行扩增与表达。基因克隆实验中常将经改造后的质粒作为运送基因的载体，质粒D2NA提取效率及质量对后续实验步骤(如酶切、连接、转化大肠杆菌与PCR扩增等)的成功与否有着直接的关系，因而高效快速地从细菌细 ...

概 述 基因组DNA的提取通常用于构建基因组文库、Southern杂交(包括RFLP)及PCR分离基因等。利用基因组DNA较长的特性，可以将其与细胞器或质粒等小分子DNA分离。加入一定量的异丙醇或乙醇，基因组的大分子DNA即沉淀形成纤维状絮团飘浮其中，可用玻棒将其取出，而小分子DNA则只形成颗粒状沉淀附于壁上及底部，从而达到提取的目的。在提取过程中，染色体会发生机械断裂，产生大小不同的片段，因此 ...