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Pcr guide Simple Fool ? s Guide P C R THE SIMPLE FOOL ? S GUIDE TO PCR version Tuesday October Version compiled by Steve Palumbi Andrew Martin Sandra Romano W Owen McMillan Ligaya Stice Gail Grabowski Department of Zoology and Kewalo Marine Laboratory Uni

Simple Fool ? s Guide P C R THE SIMPLE FOOL ? S GUIDE TO PCR version Tuesday October Version compiled by Steve Palumbi Andrew Martin Sandra Romano W Owen McMillan Ligaya Stice Gail Grabowski Department of Zoology and Kewalo Marine Laboratory University of Hawaii Honolulu HI - - FAX P C R page CTABLE OF CONTENTS Simple Fool ? s Guide P C R Table of Contents Introduction to S F G DNA Extraction For PCR General Extraction Protocol for Total DNA Variations on the Extraction Protocol The Cycle The Basic Cycle Variations in the Cycle PCR Protocols Hy gene Protocols Frequently used solutions Double-Strand DNA Ampli ?cations Double strand problems Single- Strand DNA Ampli ?cations Single strand problems Double-Strand mRNA Ampli ?cations Sequencing protocols From single strand PCR products From double strand PCR products Solid phase stripping with biotinylated primers Lambda exonuclease method Running the Sequencing Gel PCR Primers Making them Purifying them Mitochondrial primers s RNA Primers s RNA Primers Cytochrome oxidase II Primers Cytochrome b Primers D-loop Primers Cytochrome Oxidase II Primers ATPase Primers Nuclear Gene Primers bindin int creatine kinase Vector primers MtDNA maps Contributors References to Complete mtDNA Sequences Selected References to PCR Solution Appendix Abbreviations page CINTRODUCTION TO S F G Simple Fool ? s Guide P C R The Simple Fool ? s Guide to PCR a collection of PCR protocols and oligonucleotide primers is an attempt to promote sharing of PCR protocols and primer sequences from di ?erent gene regions so that redundant and costly e ?ort in the re ?nement of PCR techniques and the design and making of primers is not wasted Although PCR is extremely fast and easy there are in our minds a number of mysterious manifestations and inconsistencies that rear their ugly heads when working with new species We hope that while we might have encountered and solved some mysteries that others are running across other workers may have solved ones with which we need help Please keep in mind that these protocols are ones that we have had consistent success using on various taxa in our lab but that does not mean that they will work perfectly with the DNA with which you are working or that other methods are not better It is also not an attempt to make a complete reference for PCR techniques and primers Rather it is an attempt to collect easy PCR techniques and primers that just about anyone can use with success We have gathered some of these methods from colleagues across the country Some have been scribbled on beer-soaked coasters in dark Berkeley bars Some have resulted from midnight airport rendezvous We have found in general that people have been delightfully open and generous with their technique development and primers One of our goals is to promote continued openness with this guide The compilation of this ??guide ? is not an altruistic act We are asking that other labs share their experiences with us If you or your