VOLUME 1
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`Molecular· Cloning
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`A LABORATORY MANUAL
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`THiRD EDITION
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`www. Mo I ecu I arCI on i ng. com
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`Joseph Sambrook
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`PETER MACCALLUM CANCER INSTITUTE AND THE UNIVERSITY OF MELBOURNE, AUSTRALIA
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`David W. Russell
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`UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER, DALLAS
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`COLD SPRING HARBOR LABORATORY PRESS
`Cold Spring Harbor, New York
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`Personalis EX2160
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`Molecular Cloning
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`A LABORATORY MANUAL
`
`THIRD EDITION
`
`©2001 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
`All rights reserved
`Printed in the United States of America
`
`Front cover (paperback): The gene encoding green fluorescent protein was cloned from Aequorea victoria, a jellyfish found in abun(cid:173)
`dance in Puget Sound, Washington State. This picture of a 50-mm medusa was raken on color film by flash photography and shows light
`reflected from various morphological features of the animal. The small bright roundish blobs in the photograph are symbiotic
`amphipods living on or in the medusa. The bright ragged area in the center is the jellyfish's mouth.
`Bioluminescence from Aequorea is emitted only from the margins of the medusae and cannot be seen in this image. Bioluminescence
`of Aequorea, as in most species of jellyfish, does not look like a soft overall glow, but occurs only_at the rim of the bell and, given the right
`viewing conditions, would appear as a string of nearly microscopic fusiform green lights. The primary luminescence produced by
`Aequorea is actually bluish in color and is emitted by the protein aequorin. In a living jellyfish, light is emitted via the coupled green fluo(cid:173)
`rescent protein, which causes the luminescence to appear green to the observer.
`The figure and legend were kindly provided by Claudia Mills of the University of Washington, Friday Harbor. For further information,
`please see Mills, C.E. 1999-2000. Bioluminescence of Aequorea, a hydromedusa. Electronic Internet document available at http://faculty.
`washington.edu/cemills/Aequorea.html. Published by the author, web page established June 1999, last updated 23 August 2000.
`
`Back cover (paperback): A portion of a human cDNA array hybridized with a red fluor-tagged experimental sample and a green fluor(cid:173)
`tagged reference sample. Please see Appendix 10 for details. (Image provided by Vivek Mittal and Michael Wigler, Cold Spring Harbor
`Laboratory.)
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`Library of Congress Cataloging-in-Publication Data
`
`Sambrook, Joseph.
`Molecular cloning : a laboratory manual/ Joseph Sambrook, David W.
`Russell.-- 3rd ed.
`p.;cm.
`Includes bibliographical references and index.
`ISBN 0-87969-576-5 (cloth) -- ISBN 0-87969-577-3 (pbk)
`1. Molecular cloning--Laboratory manuals.
`[DNLM: 1. Cloning, Molecular--LaboratoryManuals. QH 440.5 Sl87m
`2001] I. Russell, David W. (David William), 1954-
`. II. Title.
`QH442.2 .S26 2001
`572.8--dc21
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`10 9 8 7 6 5 4 3 2 1
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`00-064380
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`People using the procedures in this manual do so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the
`material set forth in this manual and has no liability in connection with the use of these materials.
`
`All World Wide Web addresses are accurate to the best of our knowledge at the time of printing.
`
`Certain experimental procedures in this manual may be the subject of national or local legislation or agency restrictions. Users of this manual are responsible
`for obtaining the relevant permissions, certificates, or licenses in these cases. Neither the authors of this manual nor Cold Spring Harbor Laboratory assume
`any responsibility for failure of a user to do so.
`
`The polymerase chain reaction process and other techniques in this manual may be or are covered by certain patent and proprietary rights. Users of this man(cid:173)
`ual are responsible for obtaining any licenses necessary to ptactice PCR and other techniques or to commercialize the results of such use. COLD SPRING HAR(cid:173)
`BOR LABORATORY MAKES NO REPRESENTATION THAT USE OF THE INFORMATION IN THIS MANUAL WILL NOT INFRINGE ANY PATENT OR
`OTHER PROPRIETARY RIGHT.
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`Personalis EX2160
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`32 8
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`Introduction ~ 813
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`Inhibitors
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`oo,
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`Almost anything will inhibit PCRsifpresent in excess, ‘The common culprits include proteinase
`K (which, if given the opportunity, can degrade thermostable DNA polymerase), phenol, and
`EDTA. Other substances that can cause problems are ionicdetergents (Weyant et al. 1990),
`heparin (Beutleret al. 1990), polyanions such as spermidine (Ahokas and Erkkila 1993), hemo-
`globin, and gel-loading dyes such as bromophenolblue and xylene cyanol (Hoppeetal. 1992). In
`manycases, the chief cause oflow orerratic yields are contaminants in the template DNA, which
`is often the only componentofthe reaction supplied by the investigator. Many problems with
`PCRcan be cured simply by cleaning up the template by dialysis, ethanol precipitation, extrac-
`tion with chloroform, and/or chromatography through a suitable resin,
`
`DESIGN OF OLIGONUCLEOTIDE PRIMERS FOR BASIC PCR
`The chief goal of primer design is specificity, which is achieved only when each memberof a
`primer pair anneals in a-stable fashion to its target sequence in the template DNA. As a rule of
`thumb,the longer an oligonucleotide, the higher its specificity for a particulartarget. The follow-
`ing equations can beused to calculate the probability that a sequence exactly complementary to
`a string of nucleotides will occur by chance within a DNA sequencespacethat consists of a ran-
`dom sequence of nucleotides (Neiand Li 1979).
`K= [gj x [a —giajt
`where K is the expected frequency of occurrence within the sequence space, g is the relative G+C
`content of the sequence space, and G,C,A, and T are the numberofspecific nucleotides in the
`oligonucleotide. For a double-stranded genomeofsize N (in nucleotides), the expected number
`(n) of sites complementaryto the oligonucleotide is 1 = 2NK.
`These equations predict that an oligonucleotide of 15 nucleotides would be represented
`only once in a mammalian genome where N is ~3.0 x’ 10°. In the case of a 16-mer,thereis only —
`one chancein ten that a typical mammalian cDNAlibrary(complexity ~10’ nucleotides) will for-
`tuitously contain a sequence that exactly matchesthat of the oligonucleotide. However, these cal-
`culations are based on the assumption that the distribution of nucleotides in mammalian
`genomesis random. This is not the case because ofbias in codon usage (Lathe 1985) and because
`a significant fraction of the genomeis composed of repetitive DNA sequences and genefamilies
`(Bains 1994). To minimize problems of nonspecific annealing, it is advisable to use oligonu-
`cleotide primers longer than thestatistically indicated minimum. Because of the presence of
`repetitive elements, no more than 85% of the mammalian genome can be targeted precisely, even
`by primers that are 20 or more nucleotides in length (Bains 1994). Before synthesizing an
`oligonucleotide primer, it is prudent to scan DNA databases to check that the proposed sequence
`occurs only in the desired gene and notin vectors, undesired genes, or repetitive elements (e.g.,
`please see Mitsuhashiet al. 1994),
`Table 8-3 presents information on the design of oligonucleotide primers for basic PCR.
`Failures will be rare if the advice provided in the table is followed carefully.
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`Selecting PCR Primers
`Listed below are several steps involved in theselection of oligonucleotide primers:
`e Analysis of the target gene for potential priming sites that are free of homopolymeric tracts,
`have no obvious tendencyto form secondary structures, are not self-complementary, and have
`nosignificant homology with other sequences oneither strand of the target genome.
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`Personalis EX2160
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`Personalis EX2160
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