DNA实验技术

Construction of homemade 'T-vectors'This method is after Marchuk, D., et al., 1991, Nucl. Acids Res. 19(5), pp 1154. You will need: 10 x Taq buffer (Promega)Taq Polymerase (Promega)Phenol/chloroform mix100mM dTTPTE bufferAbsolute ethanol70% ethanol 1) Digest plasmid to be used with a blunt-e ...

一、质粒DNA的提取及鉴定　　（一）质粒DNA的提取及鉴定　　1．收获细菌　　（1）将2ml含相应抗生素的LB液体培养基加入到通气良好的15ml的试管中，接入一单菌落，于37℃剧烈振荡培养过夜。　　（2）将1.5ml培养物倒入1.5ml离心管中，用台式离心机于4℃以12000g离心5min，将剩余的培养物贮存于4℃。　　（3）吸尽培养液，使细菌沉淀尽可能干燥。　　2．碱裂解法小量抽提质粒　　（1）将细菌沉淀重悬于100μl预冷的溶液Ⅰ（50mmol/L葡萄糖，2 ...

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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 ...