Technical Tips Online最新文献

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High-resolution MADGE 高分辨率马奇
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70161-2
Xiao-He Chen, Sandra D. O'Dell, Lesley J. Hinks, Emmanuel Spanakis, Tom R. Gaunt, Rosalind H. Ganderton, Ian N.M. Day
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引用次数: 3
SmaRT-PCR: a novel application of competitive PCR for mRNA quantitation SmaRT-PCR:竞争性PCR用于mRNA定量的新应用
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70157-0
Marten Szibor, Henning Morawietz
{"title":"SmaRT-PCR: a novel application of competitive PCR for mRNA quantitation","authors":"Marten Szibor,&nbsp;Henning Morawietz","doi":"10.1016/S1366-2120(08)70157-0","DOIUrl":"https://doi.org/10.1016/S1366-2120(08)70157-0","url":null,"abstract":"<div><p>The polymerase chain reaction (PCR) was first described in 1985 (Ref. <span>[1]</span>). Since then, it has become the ‘gold standard’ for amplifying DNA and reverse-transcribed RNA. Unfortunately, the greatest advantage of PCR (its sensitivity) easily becomes its biggest disadvantage, as the smallest changes in reaction conditions from tube to tube result in large differences in the outcome.</p><p>During the past few years, there has been increasing interest in semiquantitative and quantitative PCR (Ref. <span>[2]</span>). Several protocols for how to generate and use internally deleted standards have been published (Ref. <span>3</span>, <span>4</span>, <span>5</span>). The aim of this study was to measure gene expression using the high specificity and sensitivity of competitive PCR techniques but at a lower cost.</p></div>","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"6 1","pages":"Pages 4-7"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1366-2120(08)70157-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91724463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Genetic analysis of heterozygous loci using serial single-strand conformational polymorphism analysis 利用序列单链构象多态性分析杂合位点的遗传分析
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70156-9
Bernd Kriegesmann , Bernhard Gregor Baumgartner , Bertram Brenig , Stephan Jansen
{"title":"Genetic analysis of heterozygous loci using serial single-strand conformational polymorphism analysis","authors":"Bernd Kriegesmann ,&nbsp;Bernhard Gregor Baumgartner ,&nbsp;Bertram Brenig ,&nbsp;Stephan Jansen","doi":"10.1016/S1366-2120(08)70156-9","DOIUrl":"https://doi.org/10.1016/S1366-2120(08)70156-9","url":null,"abstract":"<div><p>The detection of genetic polymorphisms is becoming increasingly important and so new methods that promote fast and efficient analysis will become necessary. In many cases, we can find individuals who are homozygous for different alleles. Evaluating the DNA sequences of these alleles is straightforward, for instance, by direct sequencing of an amplified PCR product. However, if we are looking for very rare or lethal alleles and, as a consequence, there is only a very low or even no chance of finding homozygous individuals for investigation, we have to use heterozygous individuals for genetic analysis. To avoid artefacts from direct DNA sequencing of heterozygous genomes<span><sup>1</sup></span>, the two alleles must be split by cloning them into a vector so that each allele can be investigated separately. To be certain of finding both alleles, a number of time-consuming sequencing runs must be performed. Here, we present a simple way to screen colonies of interest.</p></div>","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"6 1","pages":"Pages 1-3"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1366-2120(08)70156-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91759661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
PCR with the fluorogenic DNA stain SYBR® Green I 荧光DNA染色SYBR®Green I的PCR
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70158-2
Davidr . Johnson
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引用次数: 0
Oligonucleotide-directed mutagenesis using plasmid DNA: a screening step to confirm the first successful step to positive mutagenesis 使用质粒DNA的寡核苷酸定向诱变:确认正向诱变成功的第一步的筛选步骤
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70159-4
A. Smith
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引用次数: 0
Perfect order plating: principle and applications 完美顺序电镀:原理及应用
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70169-7
Nikita N. Khromov-Borisov , Jenifer Saffi , João A.P. Henriques
{"title":"Perfect order plating: principle and applications","authors":"Nikita N. Khromov-Borisov ,&nbsp;Jenifer Saffi ,&nbsp;João A.P. Henriques","doi":"10.1016/S1366-2120(08)70169-7","DOIUrl":"10.1016/S1366-2120(08)70169-7","url":null,"abstract":"","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"6 1","pages":"Pages 51-57"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1366-2120(08)70169-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77821803","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 12
PCR with the fluorogenic DNA stain SYBR® Green I 荧光DNA染色SYBR®Green I的PCR
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70158-2
David R. Johnson
{"title":"PCR with the fluorogenic DNA stain SYBR® Green I","authors":"David R. Johnson","doi":"10.1016/S1366-2120(08)70158-2","DOIUrl":"https://doi.org/10.1016/S1366-2120(08)70158-2","url":null,"abstract":"<div><p>SYBR® Green I is a sensitive stain for DNA in agarose gels (Ref. <span>[1]</span>). Its enormous increase in fluorescence upon binding to double-stranded DNA has led to its use in monitoring the accumulation of product during PCR (Ref. <span>2</span>, <span>3</span>). However, SYBR Green I inhibits unmodified PCR reactions. In this article, I show that using smaller amounts of SYBR Green I and supplementing the MgCl<sub>2</sub> in the reaction relieves this inhibition. In addition, PCR products labeled with SYBR Green I can be easily detected in the reaction tube with a fluorescence plate reader or in unstained agarose gels after electrophoresis.</p></div>","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"6 1","pages":"Pages 8-9"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1366-2120(08)70158-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91724464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Oligonucleotide-directed mutagenesis using plasmid DNA: a screening step to confirm the first successful step to positive mutagenesis 使用质粒DNA的寡核苷酸定向诱变:确认正向诱变成功的第一步的筛选步骤
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70159-4
Anthony M. Smith
{"title":"Oligonucleotide-directed mutagenesis using plasmid DNA: a screening step to confirm the first successful step to positive mutagenesis","authors":"Anthony M. Smith","doi":"10.1016/S1366-2120(08)70159-4","DOIUrl":"https://doi.org/10.1016/S1366-2120(08)70159-4","url":null,"abstract":"<div><p>The technique of site-directed mutagenesis (SDM) is widely used in molecular biology to introduce mutations into DNA (Ref. <span>[1]</span>). This article discusses a quick screening step that can be incorporated into mutagenesis systems such as the {GeneEditor <em>in vitro</em> site-directed mutagenesis system (Promega, Madison, WI, USA)}, which manipulates target DNA cloned into an <em>Escherichia coli</em> plasmid and uses antibiotic selection to select for mutants.</p></div>","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"6 1","pages":"Pages 10-11"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1366-2120(08)70159-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91724465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
How to prepare a DNA sequencing chromatogram for publication 如何制备用于发表的DNA测序色谱图
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70164-8
Yu Wai Chen
{"title":"How to prepare a DNA sequencing chromatogram for publication","authors":"Yu Wai Chen","doi":"10.1016/S1366-2120(08)70164-8","DOIUrl":"10.1016/S1366-2120(08)70164-8","url":null,"abstract":"","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"6 1","pages":"Pages 37-38"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S1366-2120(08)70164-8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88485222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Improved antigen detection on western blots 改进免疫印迹抗原检测
Technical Tips Online Pub Date : 2001-01-01 DOI: 10.1016/S1366-2120(08)70163-6
H. Matsugu, T. Nishikata
{"title":"Improved antigen detection on western blots","authors":"H. Matsugu, T. Nishikata","doi":"10.1016/S1366-2120(08)70163-6","DOIUrl":"https://doi.org/10.1016/S1366-2120(08)70163-6","url":null,"abstract":"","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"32 1","pages":"34-36"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89771972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
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