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PCR技术基本原理 类似于DNA的天然复制过程，其特异性依赖于与靶序列两端互补的寡核苷酸引物。PCR由变性--退火--延伸三个基本反应步骤构成： 1、模板DNA的变性：模板DNA经加热至93℃左右一定时间后，使模板DNA双链或经PCR扩增形成的双链DNA解离，使之成为单链，以便它与引物结合，为下轮反应作准备； 2、模板DNA与引物的退火(复性)：模板DNA经加热变性成单链后，温度降至55℃左右，引 ...

FocusonPCR IntroductionMore than 15 years after its initial description the polymerase chain reaction (PCR) has become a standard molecularbiology tool and is the most widely used method o ...

DNA Marker产品选择指南 1.Marker选择标准（1）应选择在目标片段大小附近ladder较密的marker，这样对目标片段大小的估计较准确。（2）所选marker应能清楚反映目标片段的大小，且次要片段大小也能反映出来。如作酶切鉴定时，目的片段和切后载体片段最好能在同一个marker中反映出来，若二者不能兼顾，将前者作为主要考虑要素。 2.常见问题分析Q：为什么marker条带非常模糊， ...

Disruption by Fusion PCR 1) In separate PCR reactions amplify the 5' and 3' ends of the gene of interest with primers about 200 bases apart. Primer 2 should begin with 24 nts complementary to t ...

Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) is an in situ method for detecting the 3'-OH ends of DNA exposed during the internucleosomal cleavage that occurs during a ...

Detection of Alu by PCR In this experiment polymerase chain reaction (PCR) is used to amplify a nucleotide sequence from chromosome 8 to look for an insertion of a short DNA sequence called Alu ...

Protocol for Paraffin Sections: Dewax paraffin sections: Incubate slides 55°C 30 min. Xylenes 2 times 2 min. each 100% EtOH 2 times 2 min. each&nb ...

Protocol I: Triton X-100 Lysis Buffer In 96 flat-wells plate incubate 4x10 6 target cells (40 wells of 105 per well) with desired concentration of effectors (105 target cells per well). After incubat ...

Designing PCR programs 1Basic Principles The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore annealing needs to take place at a suff ...

Materials from a workshop held March 1997 in Modena Italy. All materials reproduced with permission. Introduction A. Cossarizza Workshop Sponsors Methods in analysis of apoptosis and cell necrosis. Z. ...

BrdU Incorporation Protocol Day 1 Label cells with media containing100 μM BrdU (6 μL/mL of 5 mg/mL stock): Diploid Fibroblasts 4-6 hours ES cells 30 minutes 3T3 cells 2.5 hours Trypsinize ce ...

immunofluorescence labeling; that is the antibodies have the fluorescent dye attached. Direct labeling is simpler and quicker than indirect labeling. We strongly recommend direct labeling if you're pl ...

FIXATION and DNA Staining for Cell Cycle Analysis BackgroundThis method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane which allows the dye (Propidium Iodide) to enter ...

A method to re-PCR unique bands from products of mixed size INTRODUCTION The products of a PCR reaction - especially when this is done on eukaryotic genomic DNA and when using degenerate ...

Paraformaldehyde Fixation of Cells BackgroundThis fixation method is good for cells labelled by fluorochrome-conjugated antibodies to membrane antigens. It will stabilize the light scatter and labelli ...

Fixation 1) Collect 2 X 106 cells. 2) Pellet cells by spinning at 1000 rpm 4°C for 5 minutes. 3) Resuspend cell pellet in 1 ml of cold PBS. 4) Fix cells by adding 4 ml of -20°C absolute ethano ...

Colony PCR This procedure will work for both yeast and E. coli: Take a small colony and suspend it in 5ul of H2O in a PCR tube. Heat for 5 min at 95℃ and then spin the condensation down ...

Choosing/designing PCR primers In designing primers for PCR the following steps/rules were tested and proven to be useful: 1Length of individual primers between 18-24 bases. Longer primers (30-35 bp) ...

CGH of PCR Amplified Microdissected DNA PCR: We generally use 1-2 ul of starting paraffin microdissected DNA for each 50 ul DOP-PCR reaction. We assume that about 1 ug of product is produced in each D ...

停产，用次氯酸钠将房间整个清洁消毒，所有器具高温灭菌或用次氯酸钠浸泡。 地面用量：有效氯浓度2500ppm，大于30min； 器具：有效氯浓度500ppm，20min。摇床用甲醛加高锰酸钾熏蒸，用前再用紫外照射半小时。 发酵罐高温灭菌。 如果空气中噬菌体浓度也较高的话，可以用甲醛熏蒸，3g/平米；或用紫外照射，但要注意，因为这两种方法都可能对仪器造成腐蚀。下水也要用次氯酸钠处理。空气中噬菌体的检测 ...