JackD. Pollard, Jr.
Polyacrylamidegels can separate small DNA fragments (5-1000 basepairs) effectively. Resolutionand capacity of polyacrylamide gels are generally greater than agaroseones. The purified fragments can then be used for cloning, sequencing,or labeling. The protocols in this unit outline pouring and electrophoresisof nondenaturing polyacrylamide gels. Also, elution of the labeled or unlabeledseparated DNA fragments from the gels by either passive diffusion (basicprotocol) or electroelution (alternate protocols) is discussed.
Thebasic protocol describes the preparation of polyacrylamide gels for separationof small, double-stranded DNA fragments. After gel setup, DNA samples areloaded, electrophoresed through the gel, and finally purified away fromthe gel slices.
10xand 1x TBE electrophoresis buffer, pH 8.0 (APPENDIX 2)
29:1(w/w) acrylamide/bisacrylamide (see Reagents and Solutions)
10%(w/v) ammonium persulfate (in water " 1 month old, store at 4° C)
10xloading buffer (UNIT 2.5)
DNA-molecular-weightmarkers: e.g., pBR322 cut with HinfI or M13 cut with HpaII
0.5mg/ml ethidium bromide
Elutionbuffer, pH 8.0
100%and 70% ethanol
TEbuffer, pH 7.5 (APPENDIX 2)
3M sodium acetate (APPENDIX 2)
Thin-layerchromatography (TLC) plate with fluorescent indicator (e.g., Silica GelF-254 or IB-F)
Glassplates, spacers, and combs for pouring gels
Acrylamidegel electrophoresis apparatus
Syringeequipped with silanized glass wool plug (UNIT 5.6) or 2-micronfilter
BeckmanJA-20 rotor or equivalent
Additionalreagents and equipment for ethanol precipitation (Current ProtocolsUNIT2.1)
1.Assemble the gel casting apparatus.
Gelspacer and casting systems have been developed to avoid leakage. Thosewhich avoid sealing the gel with tape are best, and recently, gel castingboots that lack bottom spacers have become available (GibcoBRL). Greasingthe side /bottom spacers or pouring an agarose plug for the gel is notnecessary if some care is taken to ensure that the bottom of the plateassembly is completely sealed. Clean the gel plates thoroughly by washingthem with warm soapy water followed by an ethanol:water rinse. However,if the plates are particularly dirty or if the complete removal of anyresidual nucleic acids is required, the plates may be soaked in an 0.1M NaOH for 30 minutes prior to washing. If the gel is particularly thin(<1 mm), silanizing one or both of the plates (APPENDIX 3) facilitatespost-electrophoretic separation of the gel from the plate.
2.Prepare the gel solution (see Table 2.7.1 for appropriate acrylamide concentrationsfor resolving DNA fragments of different sizes). For a nondenaturing 5% polyacrylamide gel of 20 cm x 16 cm x 1.6 mm, 60 ml of gel solution issufficient, and it can be made by mixing the following:
6ml 10x TBE buffer
10ml of 29:1 acrylamide/bisacrylamide
44ml of water
Themigration distance (D) of double stranded DNA through a nondenaturing gelis inversely proportional to the log of its molecular weight (DÅ-log(MW)). Pick a concentration of acrylamide that will allow the desiredDNA fragments to migrate approximately one-half to three-fourths the waythrough the gel when the loading dye has reached the bottom of the gel.Also, note that the base composition of a sequence affects its electrophoreticmobility and may cause aberrant migration.
Usea flask that has a wide mouth and a spout for pouring.
Caution:Always wear gloves, safety glasses, and a surgical mask when working withacrylamide powder since it is a neurotoxin.
Commerciallyprepared polyacrylamide solutions (National Diagnostics) are availableand highly recommended since they have long shelf lives and do not involvemassing the neurotoxic acrylamide powder.
3.Vigorously agitate the solution for approximately 1 minute with magneticstirring to ensure complete mixing.
4.Add 34 ml of TEMED and swirl the flask to insure thorough mixing. Immediatelyadd 250 ml of 10 % APS and mix thoroughly. POLYMERIZATION HAS BEGUN SOALL SUCCEEDING STEPS MUST BE PERFORMED PROMPTLY. Pour the acrylamide betweenthe gel plates and insert the comb. Clamp the comb in place at the topof the gel to avoid separation of the gel from the plates as the acrylamidepolymerizes. Allow the gel to polymerize for approximately 30 minutes.
Forthick gels, pour the acrylamide directly from the mixing flask, but forthinner ones, a syringe fitted with a needle is useful. By pouring thegel slowly with a tilt 45° relative to the bench top and starting fromone corner, bubbles may be largely avoided. Also, polymerize the gel whileit is lying flat to avoid undesirable hydrostatic pressure on the gel bottom.
TEMEDmay be stored indefinitely at 4°C, but the ability of APS to efficientlyinitiate the free radical induced acrylamide polymerization diminishesgreatly over time. Make a new stock every month and store at 4°C.
Caution:Be sure to wear safety glasses while pouring the gel since splashing ofthe neurotoxic, unpolymerized acrylamide is common.
5.After polymerization is complete, remove the comb and any bottom spacersfrom the gel. Wash the gel plates free of spilled acrylamide and be surethat the spacers are properly seated and clean.
6.Fill the lower reservoir of the electrophoresis tank with 1X TBE. Initially,place the gel into the lower tank at an angle to avoid air bubbles formingbetween the plates and the gel bottom. Clamp the gel plates to the topof the electrophoresis tank and fill the upper reservoir with 1X TBE sothat the wells are covered.
Asyringe with a bent needle may be used to remove air bubbles trapped underthe gel that will disrupt the current flow.
7.Use a DC power supply to prerun and warm the gel for a least 30 minutesat 5 V/cm (constant voltage).
8.Add 10x loading buffer to DNA samples and molecular-weight markers (to1x final) and load on gel.
Loadan amount of DNA that correlates with the visualization technique to beused. If the sample is to be UV shadowed (UNIT 2.12), then 2 mg of DNAwill be required per band in a 2 cm x 2 cm x 1.6 mm well. Ethidium bromidestaining lowers the detection limit to 15 ng DNA per band. If good resolutionis desired, then only 25 mg of material should be loaded per 2 cm x 2 cmx 1.6 mm well.
Plasticdisposable pipette tips are available in a variety of styles and sizes.Choose one that fits the application. Alternatively, particularly for largervolumes, use a micropipette or pulled plastic capillary, prepared as describedin the support protocol.
9.Run the gel at about 5 V/cm, taking care to avoid excessive heating. Shorterelectrophoresis times may be achieved by running the gel at higher voltagein a cold room so long as the temperature of the gel remains below thedenaturation temperature of the sample. Run the gel until the desired resolutionhas been obtained as determined empirically or from Table 2.7.1.
From2 to 10 V/cm is acceptable. If the gel is noticeably warm to the touch,the samples in the middle will run faster or may even be denatured.
11.Turn off the power supply, and detach the gel plates from electrophoresisapparatus. Carefully pry apart the plates such that the gel is still attachedto one plate.
12.Visualize the DNA with UV shadowing (UNIT 2.12) if appropriate (sample2 mg or greater) Otherwise, stain the gel while it is still attached tothe plate for 5 to 10 min in 0.5 mg/ml ethidium bromide. If necessary,soak the gel and plate in water for 10 to 30 min to remove non-intercalatedethidium bromide and lower the background absorption.
13.Carefully wrap the gel and plate with plastic wrap. Invert and place thegel onto a UV transilluminator and photograph.
LongwaveUV light transmits through plastic wrap. Alternatively, the gel can beput directly on the transilluminator. If a photograph is not required,a longwave UV light may be shined onto the stained preparative gel to locatethe DNA fragment of interest. Avoid unnecessarily long UV exposure whichwill damage the nucleic acids. Unpolymerized acrylamide absorbs stronglyat 211 nm and may also cause shadowing that is confined to the edges andwells of the gel.
14.Cut out the desired DNA band with a scalpel or razor blade.
15.Crush the gel into many fine pieces by pushing it through a 3 ml smallbore disposable syringe to aid the diffusion of the DNA from the matrix.
Ifyou plan to use electroelution, omit this step and proceed to the alternativeprotocol.
16.Collect the pieces in an appropriately sized microcentrifuge tube.
17.Add 2 volumes elution buffer for every volume of gel. Incubate the tubewith rotation or in a shaking air incubator at room temperature.
Sinceelution is a diffusion-controlled process, more buffer will aid in elutionefficiency. Also, note that longer DNAs will take longer to diffuse fromthe gel. If speed is essential and high yields are dispensable, enoughsample can be obtained for most experiments in only a few hours of extraction.Increasing the temperature to 37°C will also speed the process. Yieldmay be increased upon repeated elutions. Small fragments (<300 basepairs)should be mostly eluted in 4 hr, but large fragments (>750 basepairs) shouldbe eluted overnight.
18.If the gel slice was cut into pieces, pellet the fragments at room temperaturefor 10 min in a tabletop centrifuge or 1 min in a microcentrifuge. Pipetteoff the supernatant solution, taking care to avoid the polyacrylamide pieces.
19.Recover any residual DNA by rinsing the gel with a small volume of elutionbuffer. Recentrifuge if necessary and combine the two supernatant solutions.
Ifnecessary, remove any remaining acrylamide pieces by filtering the supernatantthrough a syringe equipped with a disposable 0.2 micron filter.
Also,if the volume of elution buffer is too large to allow for convient precipitation,it may be reduced by successive extractions against equal volumes of butanolto concentrate the sample. About 1/5 volume of the aqueous layer is extractedinto the organic butanol layer for every volume of butanol used. If toomuch butanol is added and the water is completely extracted in the butanol,simply add more water and concentrated again.
20.Precipitate the DNA with 2 vol of 100 % ethanol by chilling for 30 minat -20°C or 10 min at -70°C. Pellet DNA by centrifuging 10 minat 12,000 x g.
Itis generally not necessary to add carrier to aid precipitation since thesmall acrylamide polymers released from the gel slice will suffice. Ifcarrier is necessary, then use either 10 mg of carrier such as tRNA orglycogen depending on the application.
21.Redissolve the DNA pellet in 100 ml TE buffer, pH 7.5, and if necessary,transfer to a microcentrifuge tube. Add 10 ml of 3 M sodium acetate, reprecipitatethe DNA with 2 vol of 100 % ethanol, and chill for 30 min at -20°Cor 10 min at -70°C. Recover the DNA by microcentrifugation as in step20.
22.Rinse the pellet twice with 70 % ethanol. After drying, the pellet maybe resuspend in TE buffer, pH 7.5, if appropriate.
|Table1 Concentrations of Acrylamide Giving Maximum Resolution of DNA Fragmentsa|
bluemarker (base pairs)
|aData are compiled from articles by Maniatis and Ptashne (1973a,b) andManiatis et al. (1975).|
Iftime is at a premium, the elution steps (15 to 19) of the basic protocolmay be replaced with the following electroelution protocol for small DNAs(<300 basepairs). Recovery of DNA fragments should be similar to thepassive elution by diffusion.
Additionalreagents and equipment for agarose gel electrophoresis (UNIT 2.5A)
1.Carry out steps 1 to 14 of the basic protocol. After cutting out the DNAband of interest without crushing, place the gel slab into a small dialysisbag with an appropriate molecular weight cutoff. Use enough 0.5x TBE bufferto surround and immerse the slab.
Thegel slabs are not crushed to allow for easy monitoring of the electroelutionprocess and to speed the elution of the DNA.
Manycommercial apparatus with less cumbersome handling procedures are alsoavailable (Schleicher & Schuell and Sialomed).
2.Place bag in a small horizontal electrophoresis apparatus containing 0.5xTBE buffer.
3.Electrophorese the DNA out of the polyacrylamide gel at ~4 V/cm acrossthe apparatus for 2 hr for small DNAs (<300 basepairs) or 6 hours forlonger DNAs.
Becauseelution times are variable and if near complete recovery is required, thegel should be UV shadowed/stained again after elution to insure that theDNA has been quantitatively removed. Should some DNA remain in the gel,
continuethe elution process.
4.Recover the DNA in the 0.5x TBE buffer. Reverse the polarity of the apparatusfor about a minute to free any bound DNA and rinse the gel slab and innersurface of dialysis bag to recover residual DNA.
5.Add 0.1 vol of 3 M sodium acetate and ethanol precipitate as describedin step 21 of the basic protocol (a second ethanol precipitation may bedone if desired).
Polyacrylamidegel electrophoresis (PAGE) offers high resolution of low-molecular-weightnucleic acids. In particular, small DNA fragments (<500 bp) that arepoorly resolved by ordinary agarose gels are easily separated on polyacrylamidegels. Depending on the pore size of the gel (3.5% to 20% polyacrylamide),a separation from 10 to 1000 bp can be achieved. The concentrations ofacrylamide that give the maximum resolution of DNA fragments have beenempirically determined as shown in Table 2.7.1.
Polyacrylamidegels have a much higher capacity for DNA than agarose gels. Up to 15 mgof material can be loaded per 2 cm x 2 cm x 1.6 mm well. This is particularlyimportant for preparation of significant amounts of small fragments. Elutionof fragments from polyacrylamide gels yields DNA that is generally devoidof contaminating material that could interfere with enzymes used in cloning,sequencing, or labeling DNA. These two qualities make polyacrylamide gelsthe preferred method for purifying significant quantities of small fragments.
Apolyacrylamide gel is formed by the polymerization of acrylamide monomersinto long chains, which are further covalently attached by a cross-linkingagent, most commonly N,N'-methylene-bisacrylamide. Polymerizationof a polyacrylamide gel is initiated by free radicals provided by ammoniumpersulfate and stabilized by TEMED. The reaction takes about 10 to 20 minto go to completion, but the reaction rate can be varied by adjusting theTEMED and ammonium persulfate concentrations.
Thepore size of a polyacrylamide gel is determined by the total percentageof acrylamide (the sum of the weights of the acrylamide monomer and cross-linker).Historically, this has been expressed as %T. For example, the 5%T gel describedabove would contain 5% (w/v) of acrylamide plus bisacrylamide. As the %Tincreases, the pore size decreases. An appropriate %T for various rangesof fragment sizes can be determined by using Table 2.7.1. The migrationdistance (D) of double stranded DNA through a nondenaturing gel is inverselyproportional to the log of its molecular weight (DÅ -log(MW)). Also,the base composition of a sequence affects its electrophoretic mobilityand may cause aberrant migration. Size markers of similar composition shouldbe used to confirm the size of the desired fragment. To separate fragmentsover a wide range of molecular weights, a pore-gradient gel can be used.In such a gel the pore size is larger at the top than at the bottom, andthe gel becomes more restrictive as the fragment runs down the gel. Suchgradient gels are difficult to pour, however, and are not commonly used.A general description of gels as electric circuits can be found in theintroduction to this chapter.
CriticalParameters and Troubleshooting
Themost important parameter for the successful separation of small DNA fragmentsby polyacrylamide gels is the polymerization reaction itself. It is importantto use only high-quality electrophoresis-grade reagents when running thegels. Acrylamide and bisacrylamide both break down in solution to acrylicacid, which affects the mobility of molecules through the gel matrix. Acrylamidesolutions should be protected from light and should not be stored for morethan a few months. Commercially prepared polyacrylamide solutions (NationalDiagnostics) are available and highly recommended since they have longshelf lives due to the incorporation of a gaseous inhibitor that preventsthe initiation of polymerization. Ammonium persulfate is stable for approximately1 month at 4°C. Clean plates are also essential in order to avoid theintroduction of bubbles into the gel when pouring.
Oneof the most common problems encountered in polyacrylamide gels is "smiling,"in which the lanes in the center of an overheated gel run faster than thelanes at the sides. This is caused by uneven dissipation of heat by thegel: the sides are cooler than the center, and samples run faster at highertemperatures. There are several ways to avoid smiling, the simplest ofwhich is to run the gel at lower voltage. An alternative is to use an apparatusthat incorporates a mechanism such as a metal plate to disperse heat evenlythroughout the gel, or an active cooling mechanism.
Inelectroelution onto DEAE membrane, the presence of a large, insoluble pelletafter recovering DNA from the membrane is probably due to acrylamide impurities.Inclusion of 0.1% SDS in the agarose minigel inhibits binding of theseimpurities. However, SDS competes weakly for DNA binding to DEAE, necessitatinga minimal electroelution time.
Becauseof the high capacity of acrylamide relative to agarose, up to 2 ug of afragment larger than 250 bp and up to 5 ug of a smaller fragment can bepurified on 2 cm x 2 cm x 1 mm lane by this method. Up to 25 mg of materialcan be purified on the larger 2 cm x 2 cm x 1.6 mm preparative gels. Afterseveral hours of shaking at 37°C, the eluted yield should be 60% to75% for larger fragment and >85% for smaller fragments. Essentially quantitativerecovery will be able to be obtained by overnight elution. Similar recoveriescan be obtained using the slightly more cumbersome electroelution alternateprotocols.
Althougha polyacrylamide gel is less convenient than an agarose gel to set up andpolymerize, this process should take <1 hr. After polymerization, gelscan be stored overnight or even for several weeks, provided precautionsare taken to prevent the drying out of the slots such as using a papertowel soaked with buffer and encased in platic wrap to keep the gel hydrated.In general, the comb should be left in the gel and the top should be coveredwith plastic wrap. The limiting factor in polyacrylamide gel electrophoresisis heating of the gel, which results in smiling. Inclusion of a mechanismto disperse the heat and/or actively cool the gel can minimize this problemand greatly shorten electrophoresis times. If either alternate protocolfor electroelution of fragments is followed, purified fragments can beobtained in <8 hr after pouring the gel.