Separation of Small DNA Fragments by Conventional Gel Electrophoresis

Transcription

1 RESOLUTION AND RECOVERY OF SMALL DNA FRAGMENTS SECTION III This section describes the use of polyacrylamide gels and sieving agarose gels for analytical or preparative separation of small double-stranded DNA fragments. Polyacrylamide gels provide somewhat better resolution as well as significantly higher capacity. Sieving agarose gels are much easier to pour and run, however, and are particularly useful for simple analytical applications. The use of denaturing polyacrylamide gels to separate single-stranded polynucleotides is described in UNITS 2.12 & 7.6. Separation of Small DNA Fragments by Conventional Gel Electrophoresis UNIT 2.7 Large amounts of small (<1000-bp) DNA fragments can be separated by conventional electrophoretic means. The purified fragments can then be used for cloning, sequencing, and labeling. In this unit, the techniques of DNA separation via both nondenaturing polyacrylamide and sieving agarose electrophoresis are discussed. Basic Protocol 1 outlines the pouring and electrophoresis of nondenaturing polyacrylamide gels. Elution of the labeled or unlabeled separated DNA fragments from the gels by either passive diffusion (see Basic Protocol 1) or electroelution (see Alternate Protocol) is described. Sieving agarose (see Basic Protocol 2) is a specially treated type of agarose designed to be used at high concentrations. Poured and run like conventional agarose gels, this matrix can resolve small DNA fragments much like a nondenaturing polyacrylamide gel. NONDENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS This protocol describes the preparation of polyacrylamide gels for separation of small double-stranded DNA fragments. After gel setup, DNA samples are loaded, electrophoresed through the gel, and finally purified away from the gel slices. Materials 10 and 1 TBE electrophoresis buffer, ph 8.0 (APPENDIX 2) 29:1 (w/w) acrylamide/bisacrylamide (see recipe; solutions are also available commercially from National Diagnostics) TEMED (N,N,N,N -tetramethylethylenediamine; store at 4 C) 10% (w/v) ammonium persulfate (APS) in water (store 1 month at 4 C) 5 loading buffer (see recipe) DNA samples DNA-molecular-weight markers: e.g., pbr322 restriction digested with HinfI or M13 digested with HpaII 0.5 µg/ml ethidium bromide Elution buffer, ph 7.5 (see recipe) 100% and 70% ethanol TE buffer, ph 7.5 (APPENDIX 2) 3 M sodium acetate, ph 5.2 (APPENDIX 2) Contributed by Joanne Chory and Jack D. Pollard, Jr. Current Protocols in Molecular Biology (1999) Copyright 1999 by John Wiley & Sons, Inc. BASIC PROTOCOL 1 Preparation and Analysis of DNA Supplement 47

2 Glass plates, spacers, and combs for casting gels Acrylamide gel electrophoresis apparatus DC power supply Thin-layer chromatography (TLC) plate with fluorescent indicator (e.g., Silica Gel F-254 or IB-F; for UV shadowing) Longwave UV transilluminator 3-ml small-bore disposable syringe Syringe equipped with silanized glass wool plug (UNIT 5.6) or 2-µm filter Centrifuge with Beckman JA-20 rotor or equivalent Additional reagents and equipment for ethanol precipitation (UNIT 2.1A) Prepare the gel 1. Assemble the gel casting apparatus. Gel spacer and casting systems have been developed to avoid leakage. Those that avoid sealing the gel with tape are best, and recently gel casting boots that lack bottom spacers have become available (Life Technologies). Greasing the side and/or bottom spacers or pouring an agarose plug for the gel is not necessary if some care is taken to ensure that the bottom of the plate assembly is completely sealed. The gel plates should be thoroughly cleaned by washing them with warm soapy water and then rinsing with 70% ethanol. However, if the plates are particularly dirty or if complete removal of any residual nucleic acids is required, the plates may be soaked in 0.1 M NaOH for 30 min prior to washing. When the gel is particularly thin (<1 mm), silanizing one or both plates (APPENDIX 3B) facilitates post-electrophoretic separation of the gel from the plate. 2. Prepare the gel solution in a flask that has a wide mouth and a spout for pouring (see Table for appropriate acrylamide concentrations for resolving DNA fragments of different sizes). For a nondenaturing 5% polyacrylamide gel of 20 cm 16 cm 1.6 mm, 60 ml of gel solution is sufficient; this can be made by mixing the following: 6 ml 10 TBE buffer 10 ml 29:1 acrylamide/bisacrylamide 44 ml H 2 O. CAUTION: Acrylamide is a neurotoxin; always wear gloves, safety glasses, and a surgical mask when working with acrylamide powder. The migration distance (D) of double-stranded DNA through a nondenaturing gel is inversely proportional to the log of its molecular weight: i.e., D log(mw). Pick a concentration of acrylamide that will allow the desired DNA fragments to have migrated approximately one-half to three-fourths of the way through the gel when the loading dye has reached the bottom of the gel. Also, note that the base composition of a sequence affects its electrophoretic mobility and may cause aberrant migration. Commercially prepared polyacrylamide solutions (National Diagnostics) are available and highly recommended, since they have long shelf lives and avoid manipulation of the neurotoxic acrylamide powder. Table Concentrations of Acrylamide Giving Maximum Resolution of DNA Fragments a Separation of Small DNA Fragments by Conventional Gel Electrophoresis Supplement 47 Acrylamide (%) Size of fragments separated (bp) Migration of bromphenol blue marker (bp) Migration of xylene cyanol marker (bp) to to to to to a Data are compiled from articles by Maniatis and Ptashne (1973a,b) and Maniatis et al. (1975). Current Protocols in Molecular Biology

3 3. Vigorously agitate the solution 1 min with magnetic stirring to ensure complete mixing. 4. Add 34 µl TEMED and swirl the flask to ensure thorough mixing. Immediately add 250 µl of 10% (w/v) APS and mix thoroughly. Pour the acrylamide between the gel plates and insert the comb. Clamp the comb in place at the top of the gel to avoid separation of the gel from the plates as the acrylamide polymerizes. Allow the gel to polymerize for 30 min. IMPORTANT NOTE: Polymerization begins as soon as APS is added to the mixture, so all succeeding actions must be performed promptly. CAUTION: Be sure to wear safety glasses while pouring the gel since splashing of the neurotoxic, unpolymerized acrylamide is common. For thick gels, the acrylamide should be poured directly from the mixing flask, but for thinner ones, a syringe fitted with a needle is useful. By pouring the gel slowly with a tilt 45 relative to the bench top and starting from one corner, bubbles may be largely avoided. Also, the gel should be polymerized lying flat to avoid undesirable hydrostatic pressure on the gel bottom. TEMED may be stored indefinitely at 4 C, but the ability of APS solution to efficiently initiate the free radical induced acrylamide polymerization diminishes greatly over time. A new APS stock should be prepared each month and stored at 4 C. Run the gel 5. After polymerization is complete, remove the comb and any bottom spacers from the gel. Wash the gel plates free of spilled acrylamide and be sure that the spacers are properly seated and clean. 6. Fill the lower reservoir of the electrophoresis tank with 1 TBE buffer. Initially, place the gel into the lower tank at an angle to avoid the formation of air bubbles between the plates and the gel bottom. Clamp the gel plates to the top of the electrophoresis tank and fill the upper reservoir with 1 TBE so that the wells are covered. A syringe with a bent needle may be used to remove air bubbles trapped under the gel, which will disrupt the current flow. 7. Use a DC power supply to prerun and warm the gel for at least 30 min at 5 V/cm (constant voltage). 8. Add 5 loading buffer to DNA samples and molecular-weight markers (to 1 final) and load on gel. Load an amount of DNA that correlates with the visualization technique to be used. If the sample is to be UV shadowed (UNIT 2.12), then 2 ìg of DNA will be required per band in a 2 cm 2 cm 1.6 mm well. With ethidium bromide staining, the detection limit is only 15 ng DNA per band; for good resolution using this method, only 25 ìg of material should be loaded per 2 cm 2 cm 1.6 mm well. Plastic disposable pipet tips are available in a variety of styles and sizes. Choose one that fits the application for use in loading the gel. Alternatively, particularly for larger volumes, use a micropipet or pulled plastic capillary, made as follows: Break a 1-ml disposable plastic (polystyrene) pipet in half and heat the center portion over a Bunsen burner until it is just soft (barely melted). Remove from heat for 1 to 2 sec and, holding vertically, quickly pull to generate a thin capillary. After the plastic has cooled, cut as desired. To calibrate, draw up a premeasured volume of water and mark capillary. 9. Run the gel at 5 V/cm, taking care to avoid excessive heating. Run the gel until the desired resolution has been obtained as determined empirically or from Table From 2 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. Preparation and Analysis of DNA Current Protocols in Molecular Biology Supplement 45

4 Shorter electrophoresis times may be achieved by running the gel at higher voltage in a cold room so long as the temperature of the gel remains below the denaturation temperature of the sample. 10. Turn off the power supply and detach the gel plates from electrophoresis apparatus. Carefully pry apart the plates so that the gel is still attached to one plate. 11a. If desired (but only for samples 2 µg), visualize the DNA with UV shadowing, using a TLC plate with a fluorescent indicator (see UNIT 2.12). 11b. Alternatively, stain the gel while still attached to the plate by submerging it for 5 to 10 min in 0.5 µg/ml ethidium bromide. If necessary, soak the gel and plate in water for 10 to 30 min to remove nonintercalated ethidium bromide to lower the background absorption. 12. Carefully wrap the gel and plate with plastic wrap. Invert, place the gel onto a UV transilluminator, and photograph. Longwave UV light transmits through plastic wrap. Alternatively, the gel can be put directly on the transilluminator. If a photograph is not required, a longwave UV light may be shined onto the stained preparative gel to locate the DNA fragment of interest. It is important to avoid unnecessarily long UV exposure, which will damage the nucleic acids. Unpolymerized acrylamide absorbs strongly at 211 nm and may also cause shadowing that is confined to the edges and wells of the gel. Separation of Small DNA Fragments by Conventional Gel Electrophoresis Supplement 45 Recover the DNA 13. Cut out the desired DNA band with a scalpel or razor blade. 14. Crush the gel into many fine pieces by pushing it through a 3-ml small-bore disposable syringe to aid the diffusion of the DNA from the matrix. If planning to use electroelution, omit this step and proceed to the Alternate Protocol. 15. Collect the pieces in an appropriately sized tube. 16. Add 2 vol elution buffer for every volume of gel. Incubate the tube with rotation or in a shaking air incubator at room temperature until desired level of elution is obtained. Since elution is a diffusion-controlled process, increasing the amount of buffer will improve elution efficiency. Also, note that larger DNAs will take longer to diffuse from the gel. If speed is essential and high yields are dispensable, enough sample can be obtained for most experiments in only a few hours of extraction. Increasing the temperature to 37 C will also speed the process. Yield may be increased upon repeated elutions. Small fragments (<300 bp) should be mostly eluted in 4 hr, but large fragments (>750 bp) will need to be eluted overnight. 17. Depending on the volume of the elution, pellet the gel fragments at room temperature for 10 min in a tabletop centrifuge or 1 min in a microcentrifuge. Pipet off the supernatant solution, taking care to avoid the polyacrylamide pieces. 18. Recover any residual DNA by rinsing the gel with a small volume of elution buffer. Recentrifuge if necessary and combine the two supernatant solutions. If necessary, remove any remaining acrylamide pieces by filtering the supernatant through a syringe equipped with a disposable 0.2-ìm filter. Also, if the volume of elution buffer is too large to allow for convenient precipitation, it may be reduced by successive extractions against equal volumes of butanol to concentrate the sample. About 1 5 vol of the aqueous layer is extracted into the organic butanol layer for every volume of butanol used. If too much butanol is added and the water is completely extracted into the butanol, simply add more water and concentrate again. Current Protocols in Molecular Biology

5 19. Precipitate the DNA by adding 2 vol of 100% ethanol and chilling 30 min at 20 C or 10 min at 70 C. Pellet DNA by centrifuging 10 min at 12,000 g. It is generally not necessary to add carrier to aid precipitation since the small acrylamide polymers released from the gel slice will suffice. If carrier is necessary, then use 10 ìg of a carrier such as trna or glycogen, depending on the application. 20. Redissolve the DNA pellet in 100 µl TE buffer, ph 7.5. If necessary, transfer to a microcentrifuge tube. Add 10 µl of 3 M sodium acetate, ph 5.2, reprecipitate the DNA with 2 vol of 100% ethanol, and chill 30 min at 20 C or 10 min at 70 C. Recover the DNA by centrifugation as in step Rinse the pellet twice with 70% ethanol. Allow pellet to dry, and resuspend in TE buffer, ph 7.5, if appropriate. ELECTROELUTION OF SMALL DNA FRAGMENTS FROM POLYACRYLAMIDE GELS When working with small DNAs (<300 bp), if time is at a premium, the passive elution by diffusion described in Basic Protocol 1 (steps 15 to 19) may be replaced with the following electroelution procedure. Recovery of DNA fragments should be similar to that obtained with passive elution. ALTERNATE PROTOCOL Additional Materials (also see Basic Protocol 1) 0.5 TBE electrophoresis buffer, ph 8.0 (APPENDIX 2) Small dialysis bag Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5A) 1. Prepare and run the gel as described in the previous protocol (see Basic Protocol 1, steps 1 to 12). Cut out the DNA band of interest but do not crush. The gel slabs are not crushed to allow easy monitoring of the electroelution process and to speed the elution of the DNA. 2. Place the gel slab in a small dialysis bag with an appropriate molecular weight cutoff, and add enough 0.5 TBE buffer to surround and immerse the slab. Many commercial electroelution apparatuses with less cumbersome handling procedures are also available (e.g., from Schleicher & Schuell and Sialomed). 3. Place bag in a small horizontal electrophoresis apparatus containing 0.5 TBE buffer. 4. Electrophorese the DNA out of the polyacrylamide gel at 4 V/cm across the apparatus for 2 hr for small DNAs (<300 bp) or 6 hr for longer DNAs. Because elution times are variable and especially if near-complete recovery is required, the gel should be UV shadowed and stained again after elution to ensure that the DNA has been quantitatively removed. Should some DNA remain in the gel, elution should be continued. 5. Recover the 0.5 TBE buffer, now containing the eluted DNA, from the electrophoresis apparatus. Reverse the polarity of the apparatus for 1 min to free any bound DNA, and rinse the gel slab and inner surface of the dialysis bag to recover residual DNA. 6. Add 0.1 vol of 3 M sodium acetate and ethanol precipitate (see Basic Protocol 1, steps 20 and 21). A second ethanol precipitation may be done if desired. Preparation and Analysis of DNA Current Protocols in Molecular Biology Supplement 45

6 BASIC PROTOCOL 2 SIEVING AGAROSE GEL ELECTROPHORESIS Sieving agarose is agarose chemically modified to give low gelling ( 35 C) and melting ( 65 C) temperatures. Sieving agarose gels are poured and run like conventional agarose gels, but resolve small DNA fragments like nondenaturing polyacrylamide gels. They finely resolve nucleic acid fragments <1000 bp and can distinguish fragments as small as 8 bp. Materials Sieving agarose (NuSieve GTG agarose) TAE or TBE electrophoresis buffer, ph 8.0 (APPENDIX 2) Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5A) and isolating DNA using low gelling/melting temperature agarose (UNIT 2.6) 1. Melt 2% to 4% sieving agarose in the appropriate buffer as outlined by the manufacturer. Pour gel of desired size for ordinary agarose gel apparatus. Sieving gels generally resolve DNA more slowly and with sharper resolution in TBE than in TAE. Smaller fragments (<300 bp) are best separated in TBE and larger ones in TAE. 2. Load sample and run gel as for an ordinary agarose gel (UNIT 2.5A). In a 4% NuSieve GTG agarose gel in TBE buffer, bromphenol blue will migrate at a rate equal to DNA fragments of <20 bp and xylene cyanol at a rate equal to 150 bp. The gel can be run with ethidium bromide (0.5 mg/ml) included; this will cause only a slight decrease in DNA mobility, due to unwinding of the helix as a result of intercalation of the dye. CAUTION: Ethidium bromide is a mutagen. 3. Isolate the fragment (see UNIT 2.6, Alternate Protocol for low gelling/melting temperature agarose, steps 2 to 9). Various manufactures have developed methods for the removal of ethidium bromide from agarose gel slices. Spin columns (Supelco) made of a negatively charged matrix that attracts ethidium bromide and repels DNA offer quick, easy handling along with high recovery. REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 4. Acrylamide/bisacrylamide 29:1 (w/w) 29 g acrylamide 1 g bisacrylamide H 2 O to 100 ml Store 1 month at 4 C CAUTION: Acrylamide is a neurotoxin. Always wear gloves when working with the unpolymerized monomer. Elution buffer, ph mm Tris Cl, ph mm NaCl 1 mm EDTA, ph 8.0 Separation of Small DNA Fragments by Conventional Gel Electrophoresis Loading buffer for nondenaturing PAGE, 5 50 mm EDTA, ph mm Tris Cl, ph % (v/v) glycerol Supplement 45 Current Protocols in Molecular Biology

7 COMMENTARY Background Information Polyacrylamide gel electrophoresis (PAGE) offers high resolution of low-molecular-weight nucleic acids. In particular, small DNA fragments (<500 bp) that are poorly resolved by ordinary agarose gels are easily separated on polyacrylamide gels. 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 of acrylamide that give the maximum resolution of DNA fragments of different sizes have been empirically determined, as shown in Table Polyacrylamide gels have a much higher capacity for DNA than agarose gels. Up to 15 µg of material can be loaded per 2 cm 2 cm 1.6 mm well. This is particularly important for preparation of significant amounts of small fragments. Elution of fragments from polyacrylamide gels yields DNA that is generally devoid of contaminating material that could interfere with enzymes used in cloning, sequencing, or labeling DNA. These two qualities make polyacrylamide gels the preferred means for purifying significant quantities of small fragments. A polyacrylamide gel is formed by the polymerization of acrylamide monomers into long chains, which are further covalently attached by a cross-linking agent, most commonly N,N -methylenebisacrylamide. Polymerization of a polyacrylamide gel is initiated by free radicals provided by ammonium persulfate and stabilized by TEMED. The reaction takes 10 to 20 min to go to completion, but the reaction rate can be varied by adjusting the TEMED and ammonium persulfate concentrations. The pore size of a polyacrylamide gel is determined by the total percentage of 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 described above would contain 5% (w/v) of acrylamide plus bisacrylamide. As the %T increases, the pore size decreases. An appropriate %T for various ranges of fragment sizes can be determined from Table The migration distance (D) of double-stranded DNA through a nondenaturing gel is inversely proportional to the log of its molecular weight that is, D log(mw). The base composition of a sequence also affects its electrophoretic mobility and may cause aberrant migration with respect to what would be expected based on size alone; hence, size markers of similar composition should be used to confirm the size of the fragment of interest. To separate fragments over 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, and becomes more restrictive as the fragment runs down the gel. Such gradient gels are difficult to pour, however, and are not commonly used. A general description of gels as electric circuits can be found in the introduction to this chapter. The unique pore size of sieving agarose allows separation of much smaller fragments than ordinary agarose. Sieving agarose gels in the range of 2% to 4% separate in the range of 8 to 1000 bp and provide an alternative to polyacrylamide gels. Although the bands on sieving gels are somewhat more diffuse and the resolution is slightly poorer, sieving gels are easier and faster to pour and run, making them useful for a variety of applications, such as checking ligation of linker monomers into ladders (UNIT 3.16). Like low gelling/melting temperature agarose, sieving agarose is used to purify DNA fragments because of its unique properties of melting at 65 C and remaining liquid at 35 C. However, DNA fragments prepared from polyacrylamide gels are generally cleaner than those prepared from sieving agarose and can be more reproducibly used as substrates for a variety of enzymes. Critical Parameters and Troubleshooting The most important parameter for the successful separation of small DNA fragments by polyacrylamide gels is the polymerization reaction itself. It is important to use only highquality electrophoresis-grade reagents when running the gels. Acrylamide and bisacrylamide both break down in solution to acrylic acid, which affects the mobility of molecules through the gel matrix. Acrylamide solutions should be protected from light and should not be stored for more than a few months. Commercially prepared polyacrylamide solutions with long shelf lives (due to the incorporation of a gaseous inhibitor that prevents the initiation of polymerization) are available (National Diagnostics) and highly recommended. Ammonium persulfate is stable for 1 month at 4 C. Clean plates are also essential in order to avoid the introduction of bubbles into the gel when pouring. Preparation and Analysis of DNA Current Protocols in Molecular Biology Supplement 45

8 Separation of Small DNA Fragments by Conventional Gel Electrophoresis Supplement 45 One of the most common problems encountered in polyacrylamide gels is smiling, in which the lanes in the center of an overheated gel run faster than the lanes at the sides. This is caused by uneven dissipation of heat by the gel: the sides are cooler than the center and samples run faster at higher temperatures. There are several ways to avoid smiling, the simplest of which is to run the gel at lower voltage. An alternative is to use an apparatus that incorporates a mechanism such as a metal plate to disperse heat evenly throughout the gel, or an active cooling mechanism. Sieving agarose does not have the integrity of regular agarose and is even more fragile than the common low gelling/melting temperature agaroses. The manufacturers recommend against using sieving agarose concentrations of <2%. Extreme care should be taken when pulling the combs out of the gel, as wells tear easily. It is usually best to remove the comb after the gel has been submerged in the electrophoresis tank. Because this type of agarose melts at 65 C, a low voltage (6 to 7 V/cm of gel) should be maintained to keep the gel from heating too much. In a 4% NuSieve GTG agarose gel in TBE buffer, bromphenol blue will migrate at a rate equal to DNA fragments of <20 bp and xylene cyanol at a rate equal to 150 bp. Anticipated Results Because of the high capacity of acrylamide relative to agarose, up to 2 µg of a fragment >250 bp and up to 5 µg of a smaller fragment can be purified on a 2 cm 2 cm 1 mm lane by the method presented in Basic Protocol 1. Up to 25 µg of material can be purified on the larger 2 cm 2 cm 1.6 mm preparative gels. After several hours of shaking at 37 C, the eluted yield should be 60% to 75% for larger fragment and >85% for smaller fragments. Essentially quantitative recovery will be obtainable by overnight elution. Similar recoveries can be obtained using the faster but slightly more cumbersome electroelution alternate method (Alternate Protocol). Yields from sieving agarsoe gels (Basic Protocol 2) are similar to those from conventional low gelling/melting temperature agarose ( 70%) as long as fragments are <1000 bp. These gels resolve best below 500 bp. Time Considerations Although a polyacrylamide gel is less convenient than an agarose gel to set up and polymerize, this process should take <1 hr in either case. After polymerization, gels can be stored overnight or even for several weeks, provided precautions are taken to prevent the slots from drying out: for instance, using a paper towel soaked with buffer and encased in plastic wrap to keep the gel hydrated. In general, the comb should be left in the gel and the top should be covered with plastic wrap. The limiting factor in polyacrylamide gel electrophoresis is heating of the gel, which results in smiling. Inclusion of a mechanism to disperse the heat and/or actively cool the gel can minimize this problem and greatly shorten electrophoresis times. If the Alternate Protocol for electroelution of fragments is followed, purified fragments can be obtained in <8 hr after pouring the gel. The sieving agarose procedure is short, especially for minigels, and can be completed in 2 hr. After the addition of ethanol to the extracted fragment, the protocol can be interrupted for as long as desired. Literature Cited Maniatis, T. and Ptashne, M. 1973a. Multiple repressor binding at the operators in bacteriophage. Proc. Natl. Acad. Sci. U.S.A. 70: Maniatis, T. and Ptashne, M. 1973b. Structure of the operators. Nature 246: Maniatis, T., Jeffrey, A., and van desande, H Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14: Key Reference Chrambach, A. and Rodbard, D Polyacrylamide gel electrophoresis. Science 172: Provides background chemistry of polymerization of acrylamide and reviews the size separation characteristics of gels with different acrylamide percentages. FMC Marine Colloids product information. Sieving agarose was developed by FMC Marine Colloids. Literature describing its properties and use is available from the company. Contributed by Joanne Chory The Salk Institute for Biological Studies La Jolla, California Jack D. Pollard, Jr. Harvard Medical School and Massachusetts General Hospital Boston, Massachusetts Current Protocols in Molecular Biology