`
`Ricinatstneediestetal
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`VOLUME 1
`
`
`
`A LABORATORY MANUAL
`
`
`
`
`
`THIRD EDITION
`
`www.MolecularCloning.com
`
`Joseph Sambrook
`
`PETER MACCALLUM CANCER INSTITUTE AND THE UNIVERSITY OF MELBOURNE, AUSTRALIA
`
`David W. Russell
`UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER, DALLAS
`
`
`
`3 COLD SPRING HARBOR LABORATORY PRESS
`Cold Spring Harbor, New York
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`Molecular Cloning
`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-
`dance in Puget Sound, Washington State. This picture of a 50-mm medusa was taken on color film by flash photography and showslight
`reflected from various morphological features of the animal. The small bright roundish blobs in the photograph are symbiotic
`amphipodsliving on or in the medusa. The bright ragged area in the centeris the jellyfish’s mouth.
`Bioluminescence from Aequorea is emitted only from the margins of the medusae and cannot be seen in this image. Bioluminescence
`ofAequorea, as in most speciesofjellyfish, does not look like a soft overall glow, but occurs onlyat the rim ofthe bell and, given the right
`viewing conditions, would appear as a string of nearly microscopic fusiform green lights. The primary luminescence produced by
`Aequoreais actually bluish in color and is emitted bythe protein aequorin.Inalivingjellyfish,light is emitted via the coupledgreen fluo-
`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, CE, 1999-2000. Bioluminescence of Aequorea, a hydromedusa. Electronic Internet documentavailable 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 cDNAarray hybridized with a red fluor-tagged experimental sample anda green fluor-
`tagged reference sample. Please see Appendix 10 for details. (Image provided by Vivek Mittal and Michael Wigler, Cold Spring Harbor
`Laboratory.)
`
`Library of Congress Cataloging-in-Publication Data
`
`Sambrook, Joseph.
`_ Molecularcloning: a laboratory manual / Joseph Sambrook, David W.
`Russell.-- 3rd ed.
`p.3.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--Laboratory Manuals. QH 440.5 $187m
`2001] I. Russell, David W. (David William), 1954-
`.
`IL. Title.
`QH442.2 S26 2001
`572.8--de21
`
`10987654321
`
`,
`
`00-064380
`
`People using the proceduresin this manual do so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respectto the
`material set forth in this manual and hasnoliability in connection with the use of these materials.
`All World Wide Web addressesare accurateto the best of our knowledge at the time ofprinting.
`
`Certain experimental proceduresin this manual may be the subject of national or locallegislation or agency restrictions. Users of this manualare 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 doso.
`The polymerase chain reaction process and other techniquesin this manual maybeor are coveredby certain patent and proprietary rights, Users of this man-
`ual are responsible for obtaining anylicenses necessary to ptactice PCR and other techniques or to commercialize the results of sach use. COLD SPRING HAR-
`BOR LABORATORY MAKES NO REPRESENTATION THAT USE OF THE INFORMATIONIN THIS MANUAL WILL NOT INFRINGE ANY PATENT OR
`OTHER PROPRIETARY RIGHT.
`
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`Soak thefilter/membranein prehybridization solution containing blocking
`agents to reduce nonspecific absorption of the radiolabeled probe.
`
`
`
`
`
`* Incubatethefilters with denatured radiolabeled
`probe under conditions that promote specific hybridization.
`
`
`
`Washthefilters under conditions that preserve nucleic acid hybrids and promote
`the dissociation of probe nonspecifically attached to thefilter or membrane.
`
`
`
`
`FIGURE 2-15 Flowchart: Sequenceof Hybridization Steps
`
`Obtain an imageofthefilter or membrane on X-rayfilm.
`Align the image with the plates and recover the
`plaques that hybridize specifically to the probe.
`
`
`
`
`Protocol 22 ge
`
`Hybridization of Bacteriophage
`DNAonFilters -
`
`FTERS CARRYING IMMOBILIZED DNA FROM PLAQUES ARE SCREENED byhybridization in situ with
`32p_labeled probes. The technique is extremely robust, highly specific, and very sensitive and
`allows the identification of a single recombinant among several thousand plaques. The plaque
`identified as hybridization-positive is then purifiedbysubsequent roundsofscreening. The over-
`all sequenceofeventsis presented inFigure 2-15. For further details onthe hybridization ofDNA
`immobilized on filters, please see Chapter6, Protocol 10.
`
` 4
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`Protocol 22: Hybridization ofBacteriophage DNA on Filters
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`MATERIALS | ns |
`
`
`CAUTION:Please see Appendix 12 for appropriate handling of materials marked with <!>.
`Buffers and Solutions
`Please see Appendix 1 for comiponenis of stock solutions, buffers, and reagents.
`Diltite stock solutions to the appropriate coicentrations.
`Chloroform <!>
`;
`Prehybridization solution <!>
`Please seé the panel on PREHYBRIDIZATION AND HYBRIDIZATION SOLUTIONSin Chapter 1,
`Protocol 32.
`
`SM
`2x SSPE
`Depending on the numberoffilters to be processed,1 liter or more mayberequired.
`Wash solution 1
`2x SSC
`0.1% (w/v) SDS
`Wash solution 2
`1x SSC
`0.1% (w/v) SDS
`Washsolution 3
`0.1% SSC
`0.1% (w/v) SDS
`Nucleic Acids and Oligonucleotides
`Filters immobilized with bacteriophage DNA
`Prepared as described in Protocol 21 ofthis chapter.
`Radiolabeled probe <!>
`Prepared as described in Chapter 9 or 10.
`
`*
`
`©
`
`Spécial Equipment
`Boiling water bath (for denaturing double-stranded probes)
`Glass (Pyrex) baking dish or other hybridization chamber
`
`ALTERNATIVE HYBRIDIZATION CHAMBERS
`Someinvestigators prefer to incubatefilters in heat-sealable plastic bags (Sears Seal-A-Meal or equiva-
`lent) (e.g., please see Chapter6, Protocol 10) during the prehybridizaton and hybridization steps, rather
`thanin crystallization dishes. The former method avoids problems with evaporation and, because the
`sealed bags can be submerged in a waterbath, ensures that the temperatures during hybridization and
`washing ate correct. The bags must be opened and resealed when changing buffers, To avoid radioac-
`tive contamination of the water bath, the resealed bag containing radioactivity should be sealed inside a
`second, noncontaminated bag,
`;
`If only a small numberoffilters are subjected to hybridization, then use a glass screw-top bottle that
`fits the rollers of a hybridization ovenin place of a crystallization dish or Seal-A-Meal bag. These bottles
`and ovens have the advantage that small volumes of hybridization solution can be used and the
`hybridization’ temperature can be accurately controlled.
`
`
`
`Gluestick, water-soluble (e.g., UHU Stic distributed by FaberCastell)
`Incubation chamberpreset at the appropriaté hybridization temperature
`Please see Step 2.
`Radioactive ink <!>°
`Radioactive ink is made by mixing a small amountof 3*P with waterproofblack drawing ink. Wefindit
`convenient to make the ink in three grades: very hot (>2000 cps on a hand-held minimonitor), hot
`(>500 cps on a hand-held minimonitor), and cool (>50 cps on a hand-held minimonitor). Use a fiber-
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`Chapter 2: Bacteriophage X and Its Vectors
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`tip pen to apply ink ofthe desired hotness to the adhesive labels. Attach radioactive-warning tape to the
`pen, andstoreit in an appropriate place.
`Reusable alternatives to radioactive ink are chemiluminescent markers available from Stratagene
`(Glogos). The markers can be used multiple times and should be exposedto fluorescentlight just prior
`to a new roundof autoradiography.
`Whatman 3MM paper
`
`
`METHOD
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`1. If thefilters are dry, float the baked or cross-linkedfilters on the surface of 2x SSPE until they
`have become thoroughly wetted from beneath. Submergethefilters for 5 minutes.
`Make sure that no air bubbles are trapped underthefilters. The filters should change from white
`to a bluish color as the aqueous solvent penetrates the poresof thefilter. Make sure that there are
`no white spots or patches remainingon thefilters before proceeding to Step 2.
`‘Transferthefilters to a Pyrex dish or other hybridization chamber containing prehybridiza-
`tion solution. Use 3 mlof prehybridization solution per 82-mm filter or 5 ml per 132-mmfil-
`ter. Incubate thefilters with gentle agitation on a rocking platform for 1-2 hours or moreat
`the appropriate temperature (i.e, 68°C when hybridization is to be carried out in aqueous
`solution; 42°C when hybridization is to be carried out in 50% formamide).
`Whatevertypeof containeris used, the important pointis thatthefilters are completely covered by the
`prehybridization solution. During prehybridization,sites on the nitrocellulosefilter that nonspecifical-
`ly bindsingle- or double-stranded DNA becomeboundbyproteinsin the blockingsolution,Agitation
`ensuresthat thefilters are continuously bathed in and evenly coated by the prehybridization fluid.
`Whetherornot to use a prehybridization solution containing formamideis largely a matter of per-
`sonalpreference. Both versions of the recommendedsolutionsgive excellent results and neither has
`clear-cut advantages over the other. However, hybridization in 50% formamideat 42°Cis less harsh
`on nitrocellulosefilters than is hybridization at 68°C in aqueoussolution. Offsetting this advantage
`is the two- to threefold slower rate of hybridization in solutions containing formamide. Nylonfil-
`ters are impervious to the deleteriouseffects of aqueous hybridization at high temperatures.
`3. Denature 22P-labeled double-stranded probes by heating for 5 minutes at 100°C. Chill the
`probetapidly in ice water. Single-stranded probes need not be denatured.
`Alternatively, the probe may be denatured by adding 0.1 volume of 3 N NaOH.After 5 minutesat
`room temperature, transfer the probeto ice water and add0.1 volume of1 M Tris-Cl (pH 7.2) and
`0.1 volume of 2.5 N HCl. After denaturation,store the probesin ice water until needed. At low tem-
`perature,the rate of reassociation of DNAis extremely slow andessentially the probe remains in a
`single-stranded form.
`Between 2 x 10° and 1 x 10° cpm of *?P-labeled probe (specific activity 5 x 10’ cpm/tg) should be
`used permilliliter of hybridization solution. Using more probe causes the background of nonspe-
`cific hybridization to increase; using less reduces the rate of hybridization.
`If radiolabeled oligonucleotides areused as probesin this step, please see Chapter 6, Protocol 10,
`or Chapter 10, Protocol 8 for hybridization and washing conditions.
`
`
`2.
`
`PURITY OF PROBES
`It is essential that probes used to screen bacteriophage >. libraries contain no sequencesthat will hybridize
`to DNA sequencesin the vector. Severalbacteriophage2 vectors, such as Agt!1, AORF8, lambda ZAP, and
`someof the 2, Charon bacteriophages, contain the E. coli lacZ gene, eitherfor immunochemical screening
`or as part of a plasmid integrated into the bacteriophage genometo assist with rescue of cloned
`sequences, oras part of the central stuffer fragment. Because DNA probesare often prepared from frag-
`ments of plasmidsthat containall orpart of the lacZ gene, even a small amountof contamination will cause
`the probeto hybridizeindiscriminately to bacteriophage plaques. To ensure that a DNA fragment does not
`contain sequences that are complementary to the vector, we recommend(i) purifying the DNA succes-
`sively through twogels (agaroseor polyacrylamide) as described in Chapter5, before carrying out theradi-
`olabeling reaction,or(ii) preparing the probe by PCR amplification and purification (please see Chapter8,
`Protocol1).
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`Protocol 22: Hybridization ofBacteriophage DNA on Filters
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`4, Add the denatured probe to the prehybridization solution covering thefilters. Incubate the
`filters for 12-16 hours at the appropriate temperature (please sce Chapter 6, Protocol 10).
`During hybridization, keep the containers holdingthefilters tightly closed to prevent the loss of
`fluid by evaporation.
`~
`To maximize the rate of annealing of the probe with its target, hybridizations are usually carried
`outin solutionsof high ionic strength (6x SSC or 6x SSPE) at a temperaturethat is 20-25°C below
`the melting temperature (please see Chapter 1, Protocols 28-30 or Chapter 6, Protocol 10). Both
`SSPE and SSC work equally well when hybridization is carried out in aqueous solvents. However,
`when formamideis included in the hybridization buffer, 6x SSPE is preferred because ofits greater
`buffering capacity. For a general discussion of the factors that affect the rate and specificity of
`hybridization of radioactive probes to nucleic acids immobilized on solid supports, please see
`Chapter 6, Protocol 10.
`:
`5. Whenthe hybridization is completed, quickly removefilters from the hybridization solution
`and immediately immerse them in a large volume (300-500 ml) of Wash solution 1 at room
`temperature.Agitate thefilters gently, turning them over at least once during washing. After
`5 minutes, transfer thefilters to a fresh batch of wash solution and continue to agitate them
`gently. Repeat the washing procedure twice more.
`At no stage during the washing procedure should thefilters be allowed to dry orto stick together.
`Only a small fraction of the probe formsspecific hybrids with the target sequences immobilized on
`the filters. Therefore, hybridization solutions may berecovered, stored, and reused for a second
`round of hybridization. However,the intensity of the signal obtained from recycled hybridization
`solution may be reduced for a variety of reasons: the reduced specific activity of the probe due to
`radioactive decay, degradation of the probe during incubation, and renaturation of double-strand-
`ed probe during thefirst hybridization step.
`‘
`6. Wash thefilters twice for 1-1.5 hours in 300-500 ml of Wash solution 2 at 68°C,
`With experience,it is possible to use a hand-held monitorto test whether washing is complete. If
`the background is still too high or if the experiment demands washing at high stringencies,
`immerse the filters for 60 minutes in 300-500 ml of Wash solution 3 at 68°C.
`,
`:
`7. Dry thefilters in the air at room temperature on sheets ofWhatman 3MM paper or stacks of
`paper towels. Streak the undersideofthefilters with a water-soluble glue stick and arrange
`the filters (numberedside up) on a clean, dry,flat sheet of 3MM paper. Firmly pressthefil-
`ters against the 3MM paper to ensure that they do not move. Apply adhesive labels marked
`with radioactive ink or chemiluminescent markers to several asymmetric locations on the
`3MM paper. These markers serve to align the autoradiograph with thefilters. Cover the fil-
`ters and labels with Saran Wrap/Cling Film. Use tape to secure the wrap to the back of the
`3MMpaperandstretch the wrap over the paper to remove wrinkles.
`8. Exposethefilters to X-ray film (Kodak XAR-2, XAR-5,or their equivalent) for 12-16 hours
`at -70°C with an intensifying screen.
`9. Develop the film andalign it with thefilters using the marksleft by the radioactive ink or flu-
`orescent marker. Use a nonradioactive red fiber-tip pen to mark the film with the positions
`of the asymmetrically located dots on the numberedfilters.
`Identify the positive plaques by aligning the orientation marks with those on the agarplate.
`When duplicate sets offilters are hybridized to the same probe,thereis less chance of confusing a
`background smudge with a positive plaque. Pick only those plaques that yield convincing
`hybridization signals on both setsoffilters for further analysis, When screening a genomiclibrary
`for a single-copy gene, expect to find no more than one positive clone per 10° plaques screened.
`When screening cDNAlibraries, the numberofpositives depends on the abundance of the mRNA
`of interest.
`,
`Filters glued to 3MM paperare readily removed in preparationfor stripping (please see Chapter 6,
`Protocol 10) by placing the 3MM paper with attached membranesin a tub of 2x SSC. The water-
`soluble glue dissolves in this solution,releasing thefilters for transfer to a stripping solution and a
`round ofhybridization to another probe,if desired,
`
`10.
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`Chapter 2: Bacteriophage X and Its Vectors
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`11. Pick each positive plaque as described in Protocol 2 andstore in 1 ml of SMcontaininga drop
`(50 wl) of chloroform.
`-
`If the alignment of the filters with the plate does not permit identification of an individual
`hybridizing plaque, an agar plug containing several plaquesin the area of interest should be cored
`using the large endofasterile Pasteur pipette. Usea fresh pipette to core each hybridization-“Posi-
`tive area.
`
`12. To purify a hybridization-positive plaque, plate an aliquot (usually 50 ul of a 10dilution)
`of the bacteriophages that are recovered from the cored agar plug and proceed with subse-
`quent rounds of screening by hybridization.
`Ideally, in a second roundplating, there should be ~300 plaques on a 100-mmplate. These plaques
`are then screened a second time by hybridization.Pick a single, well-isolated positive plaque from
`the secondary screen and subject it to additional rounds of screening’until the stock is genetically
`pure andevery plaque hybridizesto the probeofinterest. Use a plaque derived from thefinal round
`of screening to makea stock as described in Protocol’3 or4.
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`Protocol 10: Southern Hybridization ofRadiolabeled Probes to Nucleic Acids Immobilized on Membranes - 6.57
`
`
`
`
`
`wes
`
`2Blotmost ofthe liquidfrom the membranebyplacing’iton.a padof paper towels,
`“membranebetween two sheets ofSaranWrapand apply!it,
`beenremoved
`:
`3. Dry themembiane, wrap loosely iin aluminum foil or between sheets ofblottingpaper, ;
`temperature—preferablyundervacuum—until needed, To felybridizethe membran
`bridization solution and continue with Step_2of Protocol10.
`
`é recipes for. stri
`i
`re
`imersethe membrane, and treat asdescribed to
`rem
`2
`
`DFO
`
`
`
`oy mtsCl pH 8.0), TmiMEDTA
`oe eH8:0,ODIxDenhardt'sreagent
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