`
`GeneDX 1019, pg. 1
`
`

`

`Molecular
`
`Cloning
`A LABORATORY MANUAL
`SECOND EDITION
`
`All rights reserved
`© 1989 by Cold Spring Harbor Laboratory Press
`Printed in the United States of America
`
`9 8 7 6 5 4 3 2
`
`Book and cover design by Emily Harare
`
`Cover: The electron micrograph of bacteriophage A particles
`stained with uranyl acetate was digitized and assigned false color
`by computer. (Thomas R. Broker, Louise T. Chow. and James I.
`Garrels)
`
`Cataloging in Publications (lo-to
`
`Sambrook, Joseph
`Molecular cloning: a laboratory manual / E.F.
`Fritsch, T. Maniatis—an ed.
`p.
`cm.
`Bibliography: p.
`Includes index.
`ISBN 0-87969-309-6
`
`1. Molecular cloning—Laboratory manuals. 2. Eukaryotic cells-
`-Laboratory manuals. I. Fritsch, Edward F. H. Maniatis, Thomas
`111. Title.
`QH442.2.MQS 1987
`574.87’3224—dc19
`
`87-35464
`
`Researchers using the procedures of 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.
`
`Authorization to photocopy items for internal or personal use, or the internal or personal use of
`specific clients, is granted by Cold Spring Harbor Laboratory Press for libraries and other
`users registered with the Copyright Clearance Center {CBC} Transactional Reporting Service,
`provided that the base fee of $0.10 per page is paid directly to CCC. 21 Congress St, Salem MA
`01970. [0-l‘.l'i".'-lfi9-3094’5;1r 39 $00 + $0.10I This consent does not extend to other kinds of copying,
`such as copying for general distribution, for advertising or promotions] purposes, for creating
`new collective works, or for resale.
`
`All Cold Spring Harbor Laboratory Press publications may be ordered directly from Cold
`Spring Harbor Laboratory, Box 100, Cold Spring Harbor, New York 11724. Phone: 1—800-843-
`4388. In New York (516)367-8423.
`
`w.--ll
`
`GeneDX1019, p .2
`
`
`GeneDX 1019, pg. 2
`
`

`

`”IGESIING DNA WITH RESTMCIION ENZYMS
`
`Different manufacturers of restriction enzymes recommend significantly dif-
`ferent digestion conditions, even for the same restriction enzyme. Because
`most manufacturers have Optimized the reaction conditions for their particu-
`lar preparations, we recommend following the instructions on the information
`sheets supplied with the enzymes. Some manufacturers also supply concen-
`trated buffers that have been tested for efficacy with each batch of purified
`enzyme. These buffers should be used whenever possible.
`Buffers for different restriction enzymes differ chiefly in the concentration
`of NaCl that they contain. When DNA is to be cleaved with two or more
`restriction enzymes, the digestions can be carried out simultaneously if both
`enzymes work well
`in the same buffer.
`If the enzymes have different
`requirements, two alternatives are possible: (1} The DNA should be digested
`first with the enzyme that works best in the buffer of lower ionic strength.
`The appropriate amount of NaCl and the second enzyme can then be added
`and the incubation continued.
`(2) A single buffer (potassium glutamate
`buffer [KGBD can be used in which virtually all restriction enzymes work
`(Hanish and McClelland 1988; McClelland et a1. 1988:}. The dilutions of this
`buffer that are used to obtain maximal enzyme activity of various restriction
`enzymes are giVen in Table 5.7.
`
`
`
`5.28 Enzymes Used in Molecular Cloning
`
`GeneDX 1019, pg. 3
`
`
`
`GeneDX 1019, pg. 3
`
`

`

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`GeneDX 1019, pg. 4
`
`
`
`
`
`

`

`TABLE 5.7 (continued)
`
`
`
` Enzyme 0.5x 1x
`
`
`
`NcoI
`Ndel
`Nhel
`NEeIV
`Natl
`Nrul
`NsiI
`
`PfiMI
`PstI
`Poul
`PouII
`Rsal
`RerI
`Sac]
`SacII
`Sell
`SauSAI
`SauQGI
`Seal
`SchI
`SfaNI
`Sfil
`SmaI
`SnaBI
`
`35171
`Stu!
`StyI
`'1"an
`Xbal
`Xhol
`anI
`
`Bacteriophage T4 DNA
`polymerase
`E. coti DNA polymerasel
`Klenow fragment of E. coli
`DNA polymerase 1
`BacterioPhage T4 DNA
`ligasefl mMA’I‘P)
`Reverse transcriptase
`
`++
`++
`+++
`++++
`++
`++
`+
`
`++
`+++
`++
`+++
`+++
`+++
`+++
`+++
`—
`++
`++ +
`+
`+
`—
`+++
`+++
`+++
`
`++
`+
`++
`++
`+++
`++
`+++
`
`+ +
`++
`
`++
`
`++ +
`+ +
`
`+++
`+++
`+++
`++++
`++
`+++
`+
`
`+++
`+++
`+++
`+++
`+++
`+
`+++
`++
`+
`+++
`+++
`+++
`++
`+
`+++
`+++
`++
`
`++
`++
`++
`+++
`+++
`+++
`+++
`
`+ +
`++
`
`+++
`
`++
`++
`
`1.5x
`
`+++
`++
`+++
`+++
`++
`+++
`++
`
`++++
`+++
`+++
`++
`++
`-
`+
`++
`+++
`+++
`++
`+++
`++
`++
`++
`+++
`+
`
`+++
`+++
`+
`+++
`+++
`+++
`-
`
`+ + +
`+ ++
`
`++ +
`
`+
`+ +
`
`2x
`
`+++
`+
`++
`++
`++
`+++
`+++
`
`+++
`+++
`+++
`+
`+
`—-
`—
`+
`+++
`++++
`+
`++
`++
`+++
`+
`++
`—
`
`++
`+++
`+
`+
`+
`+
`—
`
`+ + +
`+++
`
`++ +
`
`+
`++
`
`[++++J Activity greater than the activity observed in buffer recommended by the manufacturer; i + ++)
`activity approximately equal to the activity observed in bufi'er recommended by the manufacturer; I + +)
`activity approximately 50—80% of that observed in buffer recommended by the manufacturer; ( +J activity
`approximately 20—50% of that observed in buffer recommended by the manufacturer; (—} activity less than
`10% of that observed in buffer recommended by the manufacturer. Bacteriophage T4 DNA polymerase and
`the Iflenow fragment of E. coh' DNA polymerase I both work well in 1.5x and 2>< KGB. Bacteriophage T4
`DNA ligaee'works well in 0.5x KGB. (Modified from McClelland et a1. 1988.]
`
`5.30 Enzymes Used in Molecular Cloning
`
`
`
`GeneDX 1019, pg. 5
`
`
`
`GeneDX 1019, pg. 5
`
`

`

`
`
`
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`GeneDX 1019, pg. 6
`
`

`

`.l 1
`
`Notes
`
`Restriction enzymes are expensive! Costs can be kept to a minimum by
`following the advice given below.
`
`- Many restriction enzymes are supplied by the manufacturer in concen-
`trated form. Often, 1 pl of many enzyme preparations is sufficient to digest
`10 pg of DNA in 1 hour. To remove small quantities of enzyme from the
`container, briefly touch the surface of the fluid with the end of a disposable
`pipette tip.
`In this way, it is possible to remove as little as 0.1 pl of the
`enzyme preparation. Alternatively. a small piece of narrow~bore plastic
`tubing (1 cm in length) can be attached to a 1-,ul Hamilton syringe and used
`to transfer a (ll-pl volume. The plastic tubing is discarded after each
`sample is pipetted.
`Concentrated solutions of restriction enzymes may be diluted immediate-
`ly before use in 1 X restriction enzyme buffer. Never dilute an enzyme in
`water, since it may denature.
`
`' Restriction enzymes are stable when they are stored at —20“C in a buffer
`containing 50% glycerol. When carrying out restriction enzyme digestions,
`prepare the reactions to the point where all reagents except the enzyme
`have been mixed. Take the enzyme from the freezer, and immediately place
`it on ice. Use a fresh, sterile pipette every time you dispense enzyme.
`Contamination of an enzyme with DNA or another enzyme can be costly
`and can create time-consuming problems. Work as quickly as possible, so
`that the enzyme is out of the freezer for as short a period of time as
`possible. Return the enzyme to the freezer immediately after use.
`
`0 Keep reaction volumes to a minimum by reducing the amount of water in
`the reaction as much as possible. However, make sure that the restriction
`enzyme contributes less than 0.1 volume of the final reaction mixture;
`otherwise, the enzyme activity may be inhibited by glycerol.
`
`the amount of enzyme can be reduced if the digestion time is
`- Often,
`increased. This can result in considerable savings when large quantities of
`DNA are cleaved. Small aliquots can be removed during the course of the
`reaction and analyzed on a minigel to monitor the progress of the digestion.
`
`0 When digesting many DNA samples with the same enzyme, calculate the
`total amount of enzyme {plus a small excess to allow for the losses involved
`in transferring several aliquots) that is needed. Remove the calculated
`amount of enzyme solution from the stock, and mix it with the appropriate
`volume of 1 X restriction enzyme bufi‘er. Dispense aliquots of the enzymef
`buffer mixture into the reaction mixtures.
`
`5.32 Enzymes Used in Molecular Cloning
`
`
`
`
`
`GeneDX 1019, pg. 7
`
`

`

`
`
`Palyacrylumide Gel Electrophoresis
`
`Acrylamide is a monomer whose structure is
`
`CH2=CH—E|2~—NH2
`o
`
`In the presence of free radicals, which are usually supplied by ammonium
`persulfate and stabilized by TEMED (.N,N,N’,N'-tetramethylethylene-
`diamine), a chain reaction is initiated in which monomers of acrylamide are
`polymerized into long chains. When the bifunctional agent NJV’-
`methylenebisacrylamide is included in the polymerization reaction,
`the
`chains become cross-linked to form a gel, whose porosity is determined by the
`length of the chains and the degree of cross-linking.
`
`T
`'r
`_ CH2:CH—leflN—CHz—N—(lf—CH=CH2
`o
`0
`
`N. N ’- methylenebisacrylamide
`
`|
`— CHE—CH—[CH2—$H-—],,CHZ— CH—[CHz—CH —]nCH2——
`|
`l
`00
`O
`O
`n
`L.
`EH2
`9%
`NH
`to
`—CH2—?H——[CHz—(fH—lnCHz—(‘JH—{CHE—$H~1nCH2—
`CO
`CO
`CO
`rlflH
`[\rlH;2
`[\IIH2
`AH:
`THz
`+H
`(f0
`(f0
`~CH2—CH—[CH2—CH—LCH2—
`
`cross~linked
`polyacrylamide
`
`The length of the chains is determined by the concentration of acrylamide in
`the polymerization reaction (between 35% and 20%): 1 molecule of cross-
`Iinker is included for every 29 monomers of acrylamide. The effective range
`of separation in nondenaturing gels containing different concentrations of
`polyacrylamide is shown in Table 6.4.
`Polyacrylamide gels are more of a nuisance to prepare and run than
`
`
`
`6.36 Gel Electrophoresis of DNA
`
`
`
`GeneDX 1019, pg. 8
`
`
`
`GeneDX 1019, pg. 8
`
`

`

`
`
`
`
`
`
`agglrose gels. They are almost always poured between two glass plates that
`are held apart by spacers and sealed by electrical tape. In this arrangement,
`most of the acrylamide solution is shielded from exposure to the air, so that
`inhibition of polymerization by oxygen is confined to a narrow layer at the top
`of the gel. Polyacrylamide gels can range in length from 10 cm to 100 cm,
`depending on the separation required; they are invariably run in a vertical
`osition. However, they have three major advantages over agarose gels: (1)
`Their resolving power is so great that they can separate molecules of DNA
`whose lengths differ by as little as 0.2% (i.e., 1 bp in 500 bp). (2) They can
`accommodate much larger quantities of DNA than agarose gels: Up to 10 pg
`of DNA can be applied to a single slot (1 cmx 1 mm) of a typical poly-
`acrylamide gel without significant loss of resolution. (3} DNA recovered from
`polyacrylamide gels is extremely pure and can be used for the most demand-
`ing purposes (e.g., microinjection of mouse embryos).
`Two types of polyacrylamide gels are in common use:
`
`
`
`1 Nondenaturing polyacrylamide gels for the separation and purification of
`fragments of double-stranded DNA. These gels are poured and run in 1 ><
`THE at low voltage (1—8 Vfcm) to prevent denaturation of small fragments
`of DNA by heat generated by the passage of electric current. Most species
`of double-stranded DNA migrate through nondenaturing polyacrylamjde
`gels at a rate that is approximately inversely proportional to the log10 of
`their size. However, their electrophoretic mobility is also affected by their
`base composition and sequence, so that DNAs of exactly the same size can
`differ in mobility by up to 10%. This effect is believed to be caused by kinks
`that form at specific sequences in double-stranded DNA. Because it is
`impossible to know whether or not the migration of an unknown DNA is
`anomalous, electrophoresis through nondenaturing polyacrylamjde gels
`cannot be used to determine the size of double-stranded DNAs.
`
`”Denaturing polyacrylamide gels for the separation and purification of
`. single-stranded fragments of DNA. These gels are polymerized in the
`presence of an agent (urea or, less frequently, formamide') that suppresses
`base pairing in nucleic acids.
`(Alkali cannot be used as a denaturing agent
`
`m 6.4 Effective Range ofSepal-ution ot‘DNAs in Polyacryluuddc
`
`
`
`
`
`
`
`Bromophenol
`Xylene
`range ofseparation
`Ewlamiie
`blueb
`cyanol FF”
`(bp)
`.~.._'
`“’1le
`100
`460
`1000—2000
`2-5
`65
`260
`30—500
`3'3
`45
`160
`60~400
`12“)
`20
`70
`40e200
`15:0
`1 5
`60
`25—1 50
`20 0
`12
`45
`6-100
`.N‘H’N
`“The hrfiffihylenebisacrylamide is included at 1;“30th the concentration of acrylamide.
`“banded erg E1ven are the approximate sizes [in nucleotide pairs} of fragments of double-
`NA with which the dye comigrates.
`
`Effective
`
`Gel Electrophoresis of DNA 6.37
`
`GeneDX 1019, pg. 9
`
`GeneDX 1019, pg. 9
`
`

`

`because it deaminates acrylamide, and methylmercuric hydroxide cannot be
`used because it inhibits polymerization.) Denatured DNA migrates through
`these gels at a rate that is almost completely independent of its base
`compOSition and sequence. Among the uses of denaturing polyacrylamide
`gels are the isalation of radiolabeled DNA probes, the analysis of the
`products of nuclease-SI digestions, and the analysis of the products of DNA
`sequencing reactions. Descriptions of denaturing poiyacrylamide gels are
`given in Chapters 11 and 13.
`
`
`
`GeneDX1019, pg. 10
`
`
`
`
`6.38 Ge},r Electrophoresis of DNA
`
`GeneDX 1019, pg. 10
`
`

`

` A
`
`Most vertical electrophoresis tanks obtained from commercial sources are
`constructed to hold glass plates 20 cm X 40 cm. However, it is possible to run
`larger or smaller gels if suitable tanks are available.
`Spacers vary in
`thickness from 0.5 mm to 2.0 min. The thicker the gel, the hotter it will
`become during electrophoresis; overheating results in “smiling” bands of
`DNA and other problems. Thinner gels are therefore preferred, since they
`produce the sharpest and flattest bands of DNA. However, it is necessary to
`use thicker gels when preparing large quantities of DNA {>1 ugfband').
`Below we describe the preparation and use of polyacrylamide gels.
`
`1. Prepare the following solutions:
`
`30% Acrylamide
`
`29 g
`1 g
`
`acrylamide
`MN'-methylenebisacrylamide
`H20 to 100 ml
`
` WHON 01" NONDENA WIRING POLYACRYMMIDE EELS
`
`Heat the solution to 37°C to dissolve the chemicals.
`
`Caution: Acrylamjde is a potent neurotoxin and is absorbed through the
`skin. The effects of acrylamide are cumulative. Wear gloves and a mask
`when weighing powdered acrylamide and methylenebisacrylamide. Wear
`gloves when handling solutions containing these chemicals. Although
`polyacrylamide is considered to be nontoxic, it should be handled with
`care because of the possibility that it might contain small quantities of
`unpolymerized acrylamide.
`
`Cheaper grades of acrylamide and bisacrylamide are often contaminated with metal
`ions. Stock solutions of acrylamide can easily be purified by stirring overnight with
`about 0.2 volume of monobed resin (MB-1, Mallinckrodt},
`followed by filtration
`through Whatman No. 1 paper.
`
`During storage, acrylamide and bisacrylamide are slowly converted to acrylic acid
`and bisacrylic acid. This deamination reaction is catalyzed by light and alkali.
`Check that the pH of the acrylamide solution is 7.0 or less. and store the solution in
`dark bottles at room temperature. Fresh solutions should be prepared every few
`months.
`
`1x TBE
`
`89 mM Tris-borate
`
`2 mm EDTA (pH 8.0)
`
`TBE is usually made and stored as a 5 x stock solution (see Table
`6.2, page 6.7}. The pH of the bufi'er should be approximately 8.3.
`
`THE is used at a working strength of 1 x for polyacrylamide gel electrophoresis.
`This is twice the strength usually used for agarose gel electrophoresis. The buffer
`reservoirs of the vertical tanks used for polyacrylamide gel electrophoresis are fairly
`small and the amount of electric current passed through them is often considerable.
`1 >< THE is required to provide adequate buffering power.
`
`
`Gel Electrophoresis of DNA 6.39
`
`GeneDX 1019, pg. 11
`
`GeneDX 1019, pg. 11
`
`

`

`10% Ammonium per-sulfate
`
`ammonium persulfate
`H20 to 10 ml
`
`1 g.
`
`The solution may be stored at 4°C for several weeks.
`
`If necessary,
`2. Prepare the glass plates and spacers for pouring the gel.
`clean them with KOHfmethanol, which is prepared by adding ~5 g of
`KOH pellets to 100 ml of methanol.
`
`Caution: Handle the KOH and the KOH!methanol solutions with great
`
`care. Use gloves and a face protector.
`
`Then wash the glass plates and spacers in warm detergent solution and
`rinse them well, first in tap water and then in deionized water. Hold the
`plates by the edges so that oils from your hands do not become deposited
`on the working surfaces of the plates. Rinse the plates with ethanol and
`set them aside to dry. The glass plates must be free of grease spots to
`prevent air bubbles from forming in the gel.
`Treat one surface of each plate with a silicone solution. This prevents
`
`spacer\
`comb \
`\.. . \
`
`back plate
`
` front plate \“x.
`
`FIGURE 6.7
`
`
`6.40 Gel Electrophoresis of DNA
`
`
`
`GeneDX1019, pg. 12
`
`
`
`GeneDX 1019, pg. 12
`
`

`

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`GeneDX 1019, pg. 13
`
`

`

`6. Wearing gloves. perform the following manipulations over a tray SO that
`any spilled acrylamide solution will not spread over the bench.
`
`a. Add 35 pl of'TEMED (N,N.N'_.N’-tetramethylethylenediamine} to each
`100 ml of acrylamide solution. Mix the solution by swirling.
`
`Invert the
`b. Draw the solution into the barrel of a 50-1111 syringe.
`syringe and expel any air that has entered the barrel.
`Introduce the
`nozzle of the syringe into the space between the two glass plates.
`Expel
`the acrylamide solution from the syringe, filling the space
`almost to the top. Keep the remaining acrylamide solution at 4°C to
`reduce the rate of polymerization.
`If the plates were clean, there
`should be no trapped air bubbles, and if they were sealed well, no
`leaks.
`If air bubbles form, empty the gel mold and repour the gel,
`after thoroughly recleaning the glass plates.
`
`c. Lay the glass plates against a test-tube rack at an angle of approxi-
`mately 10°. This decreases the chance of leakage and minimizes
`distortion of the gel.
`
`7. Immediately insert the appropriate comb, being careful not to allow air
`bubbles to become trapped under the teeth. The tOps of the teeth should
`be slightly higher than the top of the glass. Clamp the comb in place
`with a bulldog paper clip.
`If necessary, use the remaining acrylamide
`solution to fill
`the gel mold completely. Check that no acrylamide
`solution is leaking from the gel mold.
`
`8. Allow the acrylamide to polymerize for 60 minutes at room temperature.
`adding additional acrylamide solution if the gel retracts significantly.
`When polymerization is complete, a schlieren pattern will be visible just
`beneath the teeth of the comb.
`
`Gels may be stored for 1—2 days in this state before they are used. After
`polymerization is complete, surround the comb and the top of the gel with paper
`towels that have been soaked in 1 X TBE. Then seal the entire gel in Saran Wrap
`and store at 4°C.
`
`Immediately rinse out the wells with water.
`9. Carefully remove the comb.
`Using a razor blade or a scalpel, remove the electrical tape from the
`bottom of the gel.
`
`It is essential to wash out the wells thoroughly as soon as the comb is removed.
`Otherwise, small amounts of acrylamide solution trapped by the comb Wlll Hall/meg
`ice in the wells. producing irregularly shaped surfaces that give rise to dlStOI'te
`bands of DNA.
`
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`10. Attach the gel to the electrophoresis tank, using large bulldog paper “111125
`on the sides and three-prong clamps on the shoulders. The notched P a 9
`should face inward toward the buffer reservoir.
`
`.
`11. Fill the reservoirs of the electrophoresis tank With 1 x TBE. USe a hen
`
`t
`
`
`
`6.42 Gel Electrophoresis of DNA
`
`
`
`GeneDX1019, pg. 14
`
`
`
`
`
`GeneDX 1019, pg. 14
`
`

`

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`
`
`

`

`DETECTION 01‘ DNA IN POLYACRYLAMIDE EELS
`
`Staining with Ethidfltm Bromide
`
`Because polyacrylamide quenches the fluorescence of ethidium bromide, it is
`not possible to detect bands that contain less than about 10 ng of DNA by this
`method.
`
`I _
`
`Gently submerge the gel and its attached glass plate in staining solution
`{0.5 ngr’ml ethidium bromide in 1 X TBE [see page 67]). Use just enough
`staining solution to cover the gel completely. After staining for 30—45
`minutes at room temperature, remove the gel, using the glass plate as a
`support. Carefully blot excess liquid from the surface of the gel with a pad
`of Kimwipes. Do not use absorbent paper (to which the gel will stick).
`Cover the gel with a piece of Saran Wrap. Try to avoid creating air
`bubbles or folds in the Saran Wrap.
`
`Caution: Ethidium bromide is a powerful mutagen and is moderately
`toxic. Gloves should be worn when working with solutions that contain
`this dye- After use, these solutions should be decontaminated by one of
`the methods described on pages 6.16—6.17.
`
`Try to minimize the movement of the staining solution across the surface of the. gel
`during staining. The aim is to keep the gel attached to its supporting glass plate.
`If
`the gel becomes completely detached, it can usually be rescued from the staining
`solution on a large glass plate and transferred to a shallow water bath.
`In most cases,
`the gel can then be carefully unfolded and restored to its original shape. To avoid
`problems, some workers use a piece of plastic mesh (mesh size 1 cm, available from
`garden and hardware stores) to hold the gel in place during staining.
`
`To photograph the gel, place a piece of Saran Wrap on the surface of an
`ultraviolet transillumiuator.
`Invert the gel, and place it on the trans-
`illuminator. Remove the glass plate, leaving the gel attached to the Saran
`Wrap. Photograph the gel as described on page 6.19.
`
`Caution: Ultraviolet radiation is dangerous, particularly to the eyes. To
`minimize exposure, make sure that
`the ultraviolet
`light source is
`adequately shielded and wear protective goggles or a full safety mask that
`efficiently blocks ultraviolet light.
`
`6.4-1 Gel Elecb‘ophorests of DNA
`
`
`
`GeneDX 1019, pg. 16 -
`
`GeneDX 1019, pg. 16
`
`

`

`
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`
` ”v.91293a.
`
`GeneDX 1019, pg. 17
`
`

`

`ISOLATION OF DNA IRAQMENIS EROM POLYACRYLAMIDE EELS
`
`The best method to isolate DNA from polyacrylamide gels is the “crush and
`soak” technique originally described by Maxam and Gilbert (1977). The DNA
`obtained is of very high purity and is free of contaminants that inhibit
`enzymes or are toxic to transfected or microinjected cells. Although the
`method is lengthy and inefficient (<30% yield for DNA fragments > 3 kb in
`length}, it can be used to isolate both double- and single-stranded DNAs from
`neutral and denaturing polyacrylamide gels, respectively. A more rapid
`technique that is used to isolate fragments of double-stranded DNA is to
`embed the piece of polyacrylamide containing the DNA of interest into a slit
`cut in an agarose gel, and then to elute the DNA onto a sliver of DEAE-
`cellulose membrane (Schleicher and Schuell NA-45l as described on pages
`6.24—6.27.
`
`“Crush and Soak” Heated
`
`The following procedure is a modification of the technique described by
`Maxam and Gilbert (1977).
`
`1. Run the polyacrylamide gel as described on pages 6.39—6