DNA基础知识

Mini-prep1. Spin down 1.5 ml of overnight culture in eppie for 1 minute on high.2. Asprirate supernatant and resuspend cell pellet in 100 µl Solution I (25 mM Tris-HCl, pH 8.0, 10 mM EDTA).3. Add 200 µl Solution II (0.2 N NaOH, 1.0% SDS) and mix gently by inversion.4. Add 150 µl Solution III (3M KOAc, pH 4.8 ), vortex briefly to mix, a ...

Improved Alcohol Precipitation of DNAECKDescription========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 p ...

Quick DNA Plasmid Prep.This is a very fast mini-prep protocol which is suitable for sequence analysis and restriction digests. Although the yield is higher than Protocol D.1, there is considerable chromosomal DNA, RNA and protein contamination.SolutionsSucrose/Tris25% sucrose 25 g ...

Extrachromosomal elements, plasmids, selectable markersIncluding an origin of replication (i.e. the E. coli oriC region) into a circular DNA molecule is a mechanism to have an extrachromosomal element in the prokaryotic cell. Such an extrachromosomal element is called a plasmid, or vec ...

CsCl Prep of Plasmid DNAThis is a standard large scale prep. for plasmid DNA which gives a yield of 0.5 - 1.0 mg. I have made some minor changes to the MHB protocol.SolutionsSolution I, II, III from protocol D.1.Tris/EDTA pH 7.5 (optional)20 ml 1 M Tris 7.54 ml 0.5 M EDTA 8.0up to 2 liters with Qstore at 4 degrees for dialysisD ...

CaPO4 Transfection for Chromaffin Cells1. 2M CaCl2 - 11.1g CaCl2 (anhydrous) + 47.3 ml H2O (or 14.7g CaCl2-2H2O) Filter sterilize and store at 4'C 2. 150 mM Na2HPO4 -2.13g Na2HPO4 + 99.7 ml H2O (or 4.02 g Na2HPO4-7H20 + 97.8 ml Store at 4'C (pH is 9, nothing will grow In it) 3. 2x PlBS 280 mM NaC1 1.84g 40 mM PIPES 1.21g 1.5 mM Na2HPO4 1.0 ml of 1 ...

Labeling oligonucleotides with 32P ATPSteve HahnLast modified 8/13/01Wear 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 ...

AbstractGene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact ...

甲基化研究对于基因的 调控和肿瘤的发生有非常密切的关系。在一般正常的细胞中，基因调控区CpG岛处于非甲基化的状态。而当细胞发生癌变后，这些CG区域往往呈现甲基化状态。英国医学刊物《the Lancet》报道奥地利因斯布鲁克医学院的专家们早就可以通过检测大便中DNA的甲基化变化，把健康人的大便与结肠癌患者的大便区分开。大便标本中SFRP2 甲基化的程度是诊断结肠癌最灵敏的标记。研究人员发现，肿瘤的发生以及一些遗传病的出现 ...

Creating a deletion by PCR splicingContributed by Dr.A.GratchevI didn't want to place this in the methods section due to its simplicity. To delete a desired fragment from existing DNA fragment all you need is a pair of primers combined of flanking sequences and a high fidelity polymerase (a mix of Taq a ...

Whole-Genome and Custom Fine-Tiling Array CGHComparative Genomic Hybridization (CGH) measures DNA copy number differences between a reference genome and your sample genome. NimbleGen offers two high-definition array CGH products: whole-genome array CGH and fine-tiling arr ...

Nick Translation of DNA for CGH1. Prepare reaction mixtures per 50ul Add Enzymes last, gently vortex mixture, quick spin liquid to bottom of tube:LabelBiotin-dUTP Digox-dUTP FITC-dUTP Texas Red dUTP 10X Biotin dNTP5 ul 0 ul 0 ul 0 ul 10X A4 dNTP0 ul 5 ul 5 ul 5 ul dUTP0 ul 1 ul 1 ul 1 ul DNA POL-11 ul 1 ul 1 ul 1 ul DNA Pol/DNAse(additio ...

Comparative Genomic Hybridization (CGH) 上一篇：ComparativeGenomicHybridization(CGH) 下一篇：Gene-specific RT-PCR

NOTE: The protocol presented below is based on the Amplified Fragment Length Polymorphism (AFLP) technology developed by Marc Zabeau and colleagues at Keygene N.V., Agrobusiness Park 90, P.O. Box 216, NL-6700 AE Wageningen, Netherlands (Zabeau, 1992; Zabeau and Vos, 1993; Vos et al., 1995). The AFLP ...

Adsroption of Anti-E. coli Antibodies Grow E. coli strain Y1090 in L broth at 37°C overnight. Transfer to 10 mM MgSO4 and store at 4°C for future use. These cells are good for about 1 week.Soak one 132 mm nitrocellulose membrane per Petri dish in 10mM IPTG (MW=238.31). Briefly blot the excess solution from the filt ...

1)Nuclear extract: see nucprep.ptc & nucext.ptc extracts should be at least 3ug/ul Probe preparation: see endlabl.ptc & isotach.ptc. probe shouldbe 10©20k cpm/ul. Fragments larger than 400bp should not be used. Make A stock (25ug/50ul) of probe plasmid digested at one end w/ 5' overhang. 3)Binding ...

Serial Analysis of Gene Expression (SAGE)SAGE is a powerful tool that allows the analysis of overall gene expression patterns with digital analysis. Because SAGE does not require a preexisting clone, it can be used to identify and quantitate new gene as well as know genes. cDNA/AFLP (CWB Bachem)T ...

据《科学时报》2007年1月5日报道：由西北农林科技大学博士生导师王跃进教授主持完成的国家转基因植物研究与产业化开发专项“中国葡萄属植物野生种抗白粉病基因克隆”研究项目，经过6年的探索研究，近日在陕西杨凌通过了国家教育部组织的鉴定。与会专家一致认为：该课题针对世界葡萄生产中存在的问题，综合采用现代分子生物学技术，以我国特有的高抗白粉病的中国葡萄属野生种华东葡萄白河-35-1为材料，在抗白粉病基因克隆与功能分析、 ...

抗原提纯与动物免疫 　　对抗原的要求是纯度越高越好，尤其是初次免疫所用的抗原。如为细胞抗原，可取1×107个细胞作腹腔免疫。可溶性抗原需加完全福氏佐剂并经充分乳化，如为聚丙烯酰胺电泳纯化的抗原，可将抗原所在的电泳条带切下，研磨后直接用以动物免疫。　　选择与所用骨髓瘤细胞同源的BALB/c健康小鼠，鼠龄在8～12周，雌雄不限。为避免小鼠反应而不佳或免疫过程中死亡，可同时免疫3～4只小鼠。　　免疫过程和方法与多克隆抗血清制备基本相同，因动 ...

PCR克隆主要有TA克隆法, 限制性酶切与连接法，杂交法和近期开发出来的特异性重组法等。但因为TA克隆法操作最简单，快速和高效的原因，成为Taq聚合酶PCR产物的最佳克隆方法。TA克隆法由Invitrogen发明，并拥有全球TA Cloning商标的专利权。TA克隆方法（Original TA Cloning Kit）利用Taq聚合酶同时具有的末端连接酶的功能，在每条PCR扩增产物的3`端自动添加一个3`-A突出端。Invitro ...