`
`GeneDX 1017, pg. 1
`
`GeneDX 1017, 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 data
`
`Sambrook, Joseph
`Molecular cloning: a laboratory manual J E.F.
`Fritsch, T. Maniatis—2ncl ed.
`p.
`cm.
`Bibliography: p.
`Includes index.
`ISBN 0-87969—309-6
`1. Molecular cloning—Laboraton manuals. 2. Eukaryotic cells-
`vLaboratory manuals. I. Fritsch, Edward F. H. Maniatis, Thomas
`111. Title.
`198T
`QH442.2.M26
`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 {CCC} Transactional Reporting Service,
`provided that the base fee of $0.10 per page is paid directly to CCC. 21 Congress St, Salem MA
`01970. [0-37969-309-6” 89 $00 + $0.1m This consent does not extend to other kinds of copying,
`such as copying for general distribution, for advertising or promotional 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.
`
`wont-Ill
`
`
`GeneDX 1017, pg. 2
`
`GeneDX 1017, pg. 2
`
`

`

`DIGESMG DNA WITH RESTRICTIflN 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 difier 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 bufl‘er.
`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 N aCl and the second enzyme can then be added
`and the incubation continued.
`(2) A single buffer {potassium glutamate
`bufl'er [KGBD can be used in which virtually all restriction enzymes work
`(Hamish 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 Cioning
`
`GeneDX1017, pg. 3
`
`GeneDX 1017, pg. 3
`
`

`

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`
`GeneDX 1017, pg. 4
`
`GeneDX 1017, pg. 4
`
`

`

`TA BLE 5.7 (continued)
`
` Enzyme 0.524: D:
`
`
`Ncol
`++
`+++
`Ndel
`++
`+++
`Nhel
`+++
`+++
`NlaIV
`++++
`++++
`NotI
`++
`++
`Nru]
`++
`+++
`N851
`+
`+
`PfiMI
`++
`+++
`PszI
`+++
`+++
`pm]
`++
`+++
`PquI
`+++
`+++
`RsaI
`+++
`+++
`RerI
`+++
`+
`Sac]
`+++
`+++
`Sac-II
`+++
`++
`Sail
`—
`+
`Sau3AI
`++
`+++
`Say-961
`++ +
`+++
`ScaI
`+
`+++
`SchI
`+
`++
`SfflNI
`A
`+
`SfiI
`+++
`+++
`SmaI
`+++
`+++
`SnaBI
`+++
`++
`351’]
`++
`++
`Stu]
`+
`++
`StyI
`++
`++
`'1"an
`++
`+++
`Xbal
`+++
`+++
`Xhal
`++
`+++
`anl
`+++
`+++
`
`Bacteriophage T4 DNA
`polymerase
`E. coli DNA polymerasel
`Klenow fragment ofE. cob:
`DNA polymerase 1
`Bacteriophage T4 DNA
`ligase(1 mMATP)
`Reverse transcriptase
`
`+ +
`++
`
`++
`
`++ +
`+ +
`
`+ +
`++
`
`+++
`
`++
`++
`
`1.5x
`+++
`++
`+++
`+++
`++
`+++
`++
`++++
`+++
`+++
`++
`++
`—
`+
`++
`+++
`+++
`++
`+++
`++
`++
`++
`+++
`+
`+++
`+++
`+
`+++
`+++
`+++
`_
`
`+ + +
`+ ++
`
`++ +
`
`+
`+ +
`
`2x
`+++
`+
`++
`++
`++
`+++
`+++
`+++
`+++
`+++
`+
`+
`_.
`,
`+
`+++
`++++
`+
`++
`++
`+++
`+
`++
`_
`++
`+++
`+
`+
`+
`+
`,
`
`+ + +
`+ ++
`
`+ + +
`
`+
`++
`
`l++++J Activity greater than the activity observed in buffer recommended by the manufacturer; i + ++)
`activity approximately equal to the activity observed in buffer recommended by the manufacturer; I + +)
`activity approximately 50—80% of that observed in buffer recommended by the manufacturer: ( +l 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 Klenow fragment of E. coli DNA polymerase I both work well in 1.5x and 2>< KGB. Bacteriophage T4
`DNA ligase'works well in 0.5x KGB. (Modified from McClelland et a1. 1988.]
`
`5.30 Enzymes Used in Molecular Cloning
`
`
`
`
`
`GeneDX1017, pg. 5
`
`GeneDX 1017, pg. 5
`
`

`

`
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`
`GeneDX 1017, pg. 6
`
`GeneDX 1017, 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 al 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 ,uJ of the
`enzyme preparation. Alternatively. a small piece of narrow~bore plastic
`tubing (1 cm in length) can be attached to a l-ul Hamilton syringe and used
`to transfer a 0.1-n1 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 amoimt 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 enzyme}
`buffer mixture into the reaction mixtures.
`
`5.32 Enzymes Used in Molecular Cloning
`
`
`
`GeneDX 1017, pg. 7
`
`GeneDX 1017, pg. 7
`
`

`

`
`
`Polyacrylamide Gel Electrophoresis
`
`Acrylamide is a monomer Whose structure is
`
`CH2=CH—fi——NH2
`
`In the presence of free radicals, which are usually supplied by ammonium
`persulfate and stabilized by TEMIED (_MN,N‘,N’-tetraniethylethylene-
`diamine), a chain reaction is initiated in which monomers of acrylamide are
`polymerized into long chains. When the bifunctlonal agent NEW-
`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
`T
`— CH2: CH-— clfuN—CHz—N—lzlf—CH=CH2
`o
`o
`
`N. N ’-methyleneblsacrylamide
`
`|
`— CHrGH—ICHrCfHfilnCHZ—cH—[CHz—(liH—LCHE—
`(I30
`$0
`(130
`NH2
`NH
`NH2
`i”:
`NH
`cl:o
`—CH2—flirt—[CH2—?H—~]nCH2—(IJH—{CHZ—(EH—WHE—
`
`o
`
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`
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`
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`
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`+H
`$0
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`
`FIJH2
`
`cross~linked
`polyaorylamide
`
`The length of the chains is determined by the concentration of acrylamide in
`the polymerization reaction (between 3.5% and 20%): 1 molecule of cross-
`linker 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
`
`GeneDX1017, pg. 8
`
`GeneDX 1017, pg. 8
`
`

`

`
`agarose 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
`acwmmodate 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., microiujection of mouse embryos).
`Two types of polyacrylamide gels are in common use:
`
`
`
`. Nondenamring polyacrylamide gets for the separation and purification of
`fragments of double-stranded DNA. These gels are poured and run in 1 x
`TEE at low voltage (1—8 V/ cm) 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 nondenatnring polyacrylamide
`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 polyacrylamide gels
`cannot be used to determine the size of double-stranded DNAs.
`
`'Denaturing polyacrylamide gets 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
`
`
`
`6.4 Effective Range ofSeparation ofDNAs 1’n Polyacrylumide
`
`Effective
`_
`1
`Ac
`Bromophenol
`Xylene
`range ofseparation
`(9'? amuiae
`blueb
`cyanol FFh
`(bp)
`._._'
`“’1‘?“
`100
`460
`1000—2000
`2-5
`65
`260
`30—500
`3'3
`45
`160
`60a400
`12.0
`20
`70
`40—200
`15:0
`l 5
`60
`25—1 50
`20 0
`12
`45
`6-100
`.NTRN''
`“The :llffihylembisacrylamide is included at 1/ 30th the concentration of acrylamide.
`minded era given are the approximate sizes [in nucleotide pairs) of fragments of double-
`NA With which the dye comigrates.
`
`
`
`
`
`
`Gel Electrophoresis of DNA “.37
`
`GeneDX1017, pg. 9
`
`GeneDX 1017, 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 polyacryiamide
`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 polyacrylamide gels are
`given in Chapters 11 and 13.
`
`6.38 Gel Electrophoresis of DNA
`
`
`
`GeneDX1017, pg. 10
`
`GeneDX 1017, pg. 10
`
`

`

`
`
`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 mm. 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 peg/band).
`Below we describe the preparation and use of polyacrylamide gels.
`
`1. Prepare the following solutions:
`
`30% Acrylamide
`
`acrylamide
`MN'-methylenebisacrylamide
`H20 to 100 ml
`
`29 g
`1 g
`
`Heat the solution to 37°C to dissolve the chemicals.
`
`Caution: Acrylamide 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 domination 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.
`
` WHON 0F NOMENA TUBING POL YA CR I’MHIDE GEES
`
`1x TBE
`
`89 mMTrisborate
`
`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 buffer should be approximately 3.3.
`
`TBE is used at a working strength of 1 x for polyaorylamide 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 X TBE is required to provide adequate buffering power.
`
`
`Gel Electrophoresis of DNA 6.39
`
`
`
`GeneDX1017, pg. 11
`
`GeneDX 1017, 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 KGB 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
`\
`
`
`gisss rabbit ear\
`
`front plate
`
`\\\
`
`FIGURE 8.7
`
`
`6.40 Ge! Electrophoresis of DNA
`
`
`
`
`
`GeneDX1017, pg. 12
`
`GeneDX 1017, pg. 12
`
`

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`GeneDX1017, pg. 13
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`GeneDX 1017, 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 a] 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.
`
`0. 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.
`
`1—2 days in this state before they are used. After
`Gels may be stored for
`polymerization is complete, surround the comb and the top of the gel with paper
`towels that have been soaked in l 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 Will pplymer-
`ize in the wells. producing irregularly shaped surfaces that give rise to distorted
`bands of DNA.
`
`10. Attach the gel to the electrophoresis tank, using large bulldog paper “I'll-:5
`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. U59 3 be”
`
`
`t
`
`6.42 Gel Electrophoresis of DNA
`
`GeneDX1017, pg. 14
`
`GeneDX 1017, pg. 14
`
`

`

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`
`

`

`DETECTION 01‘ DNA IN POLYACBYLAMIDE EELS
`
`Staining with Ethidium 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 ,ug/ml ethidium bromide in 1 X TBE [see page 67]). Usejust 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: Ethidiurn 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 transilluminator.
`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.
`
`6.44 Gel Electrophoresis of EWA
`
`
`
`GeneDX1017, pg. 16
`
`_—___
`
`GeneDX 1017, pg. 16
`
`

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`GeneDX1017. pg. 17
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`GeneDX 1017, pg. 17
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`ISOLATION 01‘ DNA FRAGMENIS EROM POLYACRYLAMIDE GELS
`The best method to isolate DNA from polyacrylamide gels is the “crush and
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`soak” technique originally described by Maxam and Gilbert (1977}. The DNA
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`obtained is of very high purity and is free of contaminants that inhibit
`enzymes or are toxic to transfected or microinjected cells. Although the
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`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
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`neutral and denaturing polyacrylamide gels, respectively. A more rapid
`technique that is used to isolate fragments of double-stranded DNA is to
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`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-
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`cellulose membrane (Schleicher and Schuell NA-45l as described on pages
`6.24—6.27.
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`To recover