PlasmidorCosmidDNAMiniprep
This protocol can be used to isolate sufficient amount DNA from 1.5ml o/n culture or 3ml 6hr culture to do several enzyme digestions.Spin 1.5ml o/n culture at 1,2000rpm for 30 Sec. Discard the supernatant.Resuspend the pellet by vertex in 100ul QP buffer, R.T. 5min.Mix 200ul 10N NaOH and 500ul 20% SDS add water to final volume 10ml.Add 200ul the solution above to the suspension (#2), invert 5 times, R.T. 5min.Add 150......阅读全文
基因工程的载体3
⑷基因组成 lDNA至少包括61个基因，大多基因按功能相似性成簇排列，其中一部分为噬菌体生命活动的必须基因，另一部分约1／3为非必须区段。 3. l噬菌体载体的类型 插入型 （Insertion vectors ）
重组DNA的分离、克隆与测序实验手册3
G. Bacterial cell maintenanceFour strains of E. coli are used in these studies: JM101 for M13 infection and isolation (4), XL1BMRF' (Stratagene) f
How－to－build－a－BAC－library
Introduction The most important aspect of our cloning vectors is that they are based on the E. coli F-factor replicon. It allows for strict
DNA体外转染试剂_如何准备转染所需的质粒DNA
转染效率受到诸多因素的影响，除了细胞、培养基和载体等影响因素外，另外一项非常重要的因素便是DNA的质量。为了比较不同厂家的转染效率，我们分别从三家不同的供应商购买了质粒制备试剂盒来制备pEGFP-N3质粒DNA，并通过不同的方法将制备的pEGFP-N3质粒转运至NIH-3T3细胞。pEGFP-N3质
LongPCR－Reagents－and－Guidelines
Long-PCR Reagents and Guidelinesfrom George Church as Modified from Cheng et al. (1)General Guidelines for Long-PCR Conditions and Enzyme Mixtures====
DNA的酶学操作
DNA的酶学操作DNA Modifying Enzymes (Michael Blaber)Introduction to bacterial restriction/modification system. It provides very useful background knowledge
High－Efficiency－Transformation
Day 0 Make sure you have the necessary solutions (instructions for how to make them can be found here): Single-stranded carrier DNA PEG 335
质粒
Extrachromosomal Elements-Plasmids (Michael Blaber)What's plasmid? Find answer here. It provides background information on the features of plasmid
DNA转化
DNA转化Chemical Transformation· Transformation of Competent Cells (RbCl2 Method) (Goldberg Lab)Very nice protocol for E. Coli transformation inc
重组DNA的分离、克隆与测序实验手册5
C. Random fragment end-repair, size selection, and phosphorylationSince both sonicated and nebulized DNA fragments usually contain single-stranded end
Cloning－PCR－products－using－TA－vectors
Cloning PCR products using TA vectorsby Paul N. Hengen, Ph.D. *Methods and reagents is a unique monthly column that highlights current discussions in
同位素法测定底物磷酸化活性方法
实验概要Ideally, one would like to be able to directly phosphorylate substrates in an intact cell. This could potentially be performed by introducing AT
酵母转化
· Yeast Transformation (Gietz Lab)LiAc/SS-DNA/PEG Transformation· Yeast Transformation (Breeden Lab)LiAc method· Large-Scale Y
在质粒载体(plasmid－vector)中进行平末端片段的克隆
1、分别设立两个反应，用适当的限制性内切核酸酶消化1－10μg质粒DNA和外源DNA片段，使它们能产生平末端。2、苯酚：氯仿抽提与乙醇沉淀法纯化出已被消化的载体和外源DNA片段。3、分别用TE（pH 8.0）重新溶解纯化出的两种DNA沉淀，使终浓度为100 ng/ml。假设1 bp相当于660Da，
细胞遗传学——原位杂交(ISH)
In Situ Hybridization· In Situ Hybridization (jsmith1@po-box.mcgill.ca)In situ hybridization, as the name suggests, is a method of localizing,
Screening－Bacterial－Colonies－Using－Xgal－and－IPTG:－αComplementation
实验概要 α-complementation occurs when two inactive fragments of E. coli β-galactosidase associate to form a functional enzyme. Many plasmid
DNA的诱变和甲基化
· In Vitro Mutagenesis Using Altered Sites (Bowtell Lab) In vitro Mutagenesis with dut ung single stranded DNA (Hahn Lab)· Site-direct
黏粒的定义组成和功能
黏粒英文cosmid，指带有黏端位点(cos)的质粒。黏粒是由人工构建的含有λ噬菌体DNA的cos序列和质粒复制子的载体。黏粒的组成包括质粒复制起点（ColE1）、氨苄青霉素抗性标记、cos位点。
Fast－Yeast－Transformation
Protocol: Fast yeast transformation Add 50 µl carrier DNA to a 1.5 ml tube. scrap cells from plate and add to the carrier DNA. Add in the fo
Construction－and－Manipulation－of－LargeInsert－Bacterial－Clone－Libraries
Acknowledgements The organizer of the workshop acknowledges Dr. Murray Milford, Professor and Interim Head, and Dr. Mark Hussey, Professor and Interim
致瘤质粒的定义
中文名称致瘤质粒英文名称tumor-inducing plasmid;tumor induction plasmid定 义致瘤农杆菌(Agrobacterium tumefaciens)中的一种大的接合型质粒。菌体感染植物伤口时，致瘤质粒可转移入被感染的植株，致瘤质粒的一段DNA(T-DNA)能插
同位素法测定底物磷酸化活性方法－Phosphorylation－of－Substrates
Phosphorylation of SubstratesScott T. Eblen, N. Vinay Kumar, and Michael J. WeberDepartment of Microbiology and Cancer Center, University of Virginia
基因工程的载体和工具酶3
2.M13噬菌体载体的构建 ⑴ 在IS区内插入LacZ基因 ⑵在标记基因区内组装MCS区段 所以能通过a互补在X-Gal/ IPTG平板上识别重组体。这类载体包括了 M13mp8、9 和 M13mp18 、 19等 这类载体的突出优点在于其既可以提供单链DNA，也可以提供双链的D
Long－PCR－Reagents－and－Guidelines
George Church Lab, Harvard Medical SchoolPCR_protocol.html">http://twod.med.harvard.edu/labgc/estep/longPCR_protocol.htmlEfficient Long PCR results fr
L.－acidophilus－transformation
OverviewElectrotransformation procedure for Lactobacillus acidophilusProcedurePrepare Electrocompetent cellsInoculate overnight culture at 10^6 CFU/ml
DNA重组（DNA－recombination）技术：DNA重组的载体2
二、噬菌体载体作为细菌寄生物的噬菌体，大多数具有编码多种蛋白质的基因，能利用宿主细胞的蛋白质合成体系，进行生长和增殖。构建的噬菌体载体，以λ噬菌体、M13和粘粒最为常用。㈠ λ噬菌体载体野生型λDNA是一种基因组为4.8 kb的线性双链DNA，全部序列已知，共编码50多个基因。其中约一半基因参与
质粒是什么
质粒（plasmid）是细菌、酵母菌和放线菌等生物中染色体（或拟核）以外的DNA分子，存在于细胞质中，具有自主复制能力，使其在子代细胞中也能保持恒定的拷贝数，并表达所携带的遗传信息，是闭合环状的双链DNA分子。质粒不是细菌生长繁殖所必需的物质，可自行丢失或人工处理而消除，如高温、紫外线等。质粒携带的
质粒是什么
质粒（plasmid）是细菌、酵母菌和放线菌等生物中染色体（或拟核）以外的DNA分子，存在于细胞质中，具有自主复制能力，使其在子代细胞中也能保持恒定的拷贝数，并表达所携带的遗传信息，是闭合环状的双链DNA分子。质粒不是细菌生长繁殖所必需的物质，可自行丢失或人工处理而消除，如高温、紫外线等。质粒携带的
质粒是什么
质粒（plasmid）是细菌、酵母菌和放线菌等生物中染色体（或拟核）以外的DNA分子，存在于细胞质中，具有自主复制能力，使其在子代细胞中也能保持恒定的拷贝数，并表达所携带的遗传信息，是闭合环状的双链DNA分子。质粒不是细菌生长繁殖所必需的物质，可自行丢失或人工处理而消除，如高温、紫外线等。质粒携带的
Agrobacterium－growth－and－transformation
Growth and storage of Agrobacterium tumefaciensStrain GV3101: resistant to gentamycin and rifampicin so add 25-50 ug/ml Gentamycin, 10 ug/ ml rifampic