Xiao-He Chen, Sandra D. O'Dell, Lesley J. Hinks, Emmanuel Spanakis, Tom R. Gaunt, Rosalind H. Ganderton, Ian N.M. Day
{"title":"High-resolution MADGE","authors":"Xiao-He Chen, Sandra D. O'Dell, Lesley J. Hinks, Emmanuel Spanakis, Tom R. Gaunt, Rosalind H. Ganderton, Ian N.M. Day","doi":"10.1016/S1366-2120(08)70161-2","DOIUrl":"https://doi.org/10.1016/S1366-2120(08)70161-2","url":null,"abstract":"","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"6 1","pages":"15-23"},"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)70161-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90126190","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}
{"title":"SmaRT-PCR: a novel application of competitive PCR for mRNA quantitation","authors":"Marten Szibor, 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}
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 , Bernhard Gregor Baumgartner , Bertram Brenig , 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}
{"title":"PCR with the fluorogenic DNA stain SYBR® Green I","authors":"Davidr . Johnson","doi":"10.1016/S1366-2120(08)70158-2","DOIUrl":"https://doi.org/10.1016/S1366-2120(08)70158-2","url":null,"abstract":"","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"15 1","pages":"8-9"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86667863","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}
{"title":"Oligonucleotide-directed mutagenesis using plasmid DNA: a screening step to confirm the first successful step to positive mutagenesis","authors":"A. Smith","doi":"10.1016/S1366-2120(08)70159-4","DOIUrl":"https://doi.org/10.1016/S1366-2120(08)70159-4","url":null,"abstract":"","PeriodicalId":101210,"journal":{"name":"Technical Tips Online","volume":"35 1","pages":"10-11"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89730706","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}
{"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}
{"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}
{"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}
{"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}