D GENE™ Denaturing Gel Electrophoresis System Instruction Manual and Applications Guide Catalog Numbers 170-9000 through 170-9070 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
Warranty The D GENE lid, tank, casting stand, gradient mixer, and accessories are warranted against defects in materials and workmanship for 1 year. If any defects occur in the instruments or accessories during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts at its discretion without charge. The following defects, however, are specifically excluded: 1. Defects caused by improper operation. 2.
Table of Contents Page Section 1 Equipment Overview ..................................................................................1 1.1 1.2 1.3 Safety .........................................................................................................................1 Specifications .............................................................................................................2 Description of Major Components ........................................................................
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Section 1 Equipment Overview 1.1 Safety Read the manual before using the D GENE system. For technical assistance, contact your local Bio-Rad Office or, in the U.S., call technical services at 1-800-4BIORAD (1-800-4246723). This instrument is intended for laboratory use only. This product conforms to the “Class A” standards for electromagnetic emissions intended for laboratory equipment applications.
2. Do not touch any wet surface before all the electrical sources are disconnected. 3. To allow maximum heat dissipation, do not put anything on the top surface of the cover. Important precautions after the run 1. Always turn off power switches and unplug all cables to DC and AC sources. Allow the heater tube to cool down (more than 15 seconds) before removing it from the tank. The ceramic tube may be very hot after shut down. Do not touch the ceramic tube for several minutes after turning off the power. 2.
DC Power Requirement External DC voltage power supply. This power supply must be ground isolated in such a way that the DC voltage output floats with respect to ground. Maximum voltage limit 300 VDC Maximum power limit 50 W Size and Weight Overall size Lid and Tank Assembly: 39 cm (L) x 20 cm (W) x 42 cm (H) Shipping weight 16 Kg Environmental Requirements Storage environment 0–70 °C, humidity 0–95% (non-condensing) Operating environment 0–35 °C, humidity 0–95%.
the core to provide a greaseless, leak-free seal for the upper buffer. Each sandwich forms one side of the cathode chamber. If only one gel is to be run, use a set of glass plates without a spacer to form a buffer dam sandwich. In addition, the core has built-in passage for upper and lower buffer circulation by the pump. 4. Comb Gasket Holder The comb gasket holder seals the top edge of the glass sandwich for casting perpendicular gradient gels.
Section 2 Introduction to Technology 2.1 Overview of Denaturing Gradient Gel Electrophoresis There is an increasing need for practical, efficient, and inexpensive ways to identify mutations responsible for genetic diseases and cancer development. PCR has solved the problem of the target limitation. When the precise site of a point mutation is not known, it is necessary to first determine the region harboring the defect.
Denaturant 0% 100% Partially melted “mutant” Partially melted “wild type” Single strands Electrophoresis * Wild Type * Mutant Double strand Fig. 2.1. An example of DNA melting properties in a denaturing gradient gel. At a low concentration of denaturants the DNA fragment remains double stranded, but as the concentration of denaturants increases the DNA fragment begins to melt. Then, at very high concentrations of denaturants, the DNA fragment can completely melt, creating two single strands.
Section 3 DGE Background Information 3.1 Introduction In DGGE, DNA is melted by using chemicals denaturants and increased temperature. A solution of 100% chemical denaturant consists of 7 M urea and 40% formamide. Denaturing gels are typically run at temperatures between 50 and 65 °C. The size of the DNA fragments run on a denaturing gel can be as large as 1 kb in length, but only the lower melting domains will be available for mutation analysis.
Wild Type DNA Mutant DNA Denature and reanneal Homoduplex DNA Heteroduplex DNA CDGE Gel wt mut wt + mut Heteroduplexes Homoduplexes Fig. 3.1. An example of wild-type and mutant DNA fragments that were denatured and re-annealed to generate four fragments, two heteroduplexes and two homoduplexes. The melting behavior of the heteroduplexes is altered so that they melt at a lower denaturant concentration than the homoduplexes, and can be visualized on a denaturing gel, for example a CDGE gel. 3.
presence of two or more melting domains. This is caused by the melting of the first domain which leads to an almost complete stop in the gel migration. This can be resolved by increasing the run time so that higher melting domains can be seen. Fig. 3.2. Perpendicular denaturing gradient gel in which the denaturing gradient is perpendicular to the electrophoresis direction.
denaturant gels usually take about 2–6 hours to reach good resolution between mutant and wild-type DNA fragments. An example of parallel DGGE is shown in Figure 3.4. In parallel DGGE, separation is not time dependent for optimal separation. This is due to the fact that a molecule will migrate with a constant velocity until it reaches the position in the gel where the denaturant concentration causes the molecule to begin to melt.
In constant denaturing gels, only a single denaturing condition is used in the gel. With the constant denaturing gels, it is possible to run more samples under the optimal conditions. Gel combs are used to form wells in the gel and depending on the number of samples being run, different combs with different numbers of wells can be used. Constant denaturing gels usually take about 2–4 hours to reach good resolution between mutant and wild-type DNA fragments.
Section 4 Sample and Reagent Preparation 4.1 Sample Preparation In some cases, adding a high melting domain to the DNA fragment allows one to analyze mutations that normally are not seen.210, 211 The addition of a 30–40 base pair GC clamp to the DNA fragment during PCR creates a high melting domain and will influence the other melting domains.196 As a result, the sequence of interest should be in the first (low) melting domain and therefore, can be analyzed.
50x TAE Buffer (1 L) Tris base 242.0 g Acetic acid, glacial 57.1 ml 0.5 M EDTA, pH 8.0 100.0 ml Mix and add dH2O to 1 L. Autoclave for 20–30 minutes. Store at room temperature. 0% Denaturing Solution (100 ml) 7.5% Gel 10% Gel 12.0% Gel 40% Acrylamide/Bis (37.5:1) 18.8 ml 25.0 ml 30.0 ml 50x TAE buffer 2.0 ml 2.0 ml 2.0 ml dH2O to 100 ml to 100 ml to 100 ml Degas for about 10–15 minutes. Store at 4 °C in a brown bottle for about 1 month. 100% Denaturing Solution (100 ml) 7.5% Gel 10% Gel 12.
dH2O 6860.0 ml 4.3 Gel Volumes The final gel volumes to use for the three different gel sizes are listed below. Spacer Thickness 0.75 mm 1.00 mm 1.50 mm 16 x 16 cm gel 25 ml 30 ml 45 ml 16 x 10 cm gel 15 ml 20 ml 26 ml 7.5 x 10 cm gel* 8 ml (16 ml) 10 ml (20 ml) 14 ml (24 ml) * The first volume is for one gel. The volume in parenthesis is for two gels.
Section 5 Buffer Temperature 5.1 Temperature Controller The temperature controller maintains the desired buffer temperature in the D GENE system (Figure 5.1). The actual and set buffer temperatures are displayed in °C. The set temperature can be adjusted by using the raise and lower buttons. The maximum buffer temperature that can be set is 65 °C. The heater light will come on when the heater is on. ACTUAL HEATER SET Fig. 5.1. The Temperature Controller displays the actual and set temperatures. 5.
Section 6 Gel Casting 6.1 Assembling the Glass Plate Sandwiches To insure proper alignment, make sure all plates and spacers are clean and dry before assembly. Use caution when assembling the glass plate sandwiches. Wear gloves and eye protection. 1. Assemble the gel sandwich on a clean surface. Lay the long rectangular plate down first, then place the left and right spacers of equal thickness along the short edges of the rectangular plate.
Fig. 6.2. Adapting the clamps to the glass plate assembly. 5. Place the sandwich assembly in the alignment slot of the casting stand (the alignment slot is the slot without cams) with the short glass plate forward (Figure 6.3). Loosen the sandwich clamps and insert a D GENE alignment card to keep the spacers upright. Note: Always use the alignment slot and alignment card to set the spacers in place.
7. Remove the alignment card. Remove the sandwich assembly from the casting stand and check that the plates and spacers are flush at the bottom. If the spacers and glass plates are not flushed, realign the sandwich and spacers to obtain a good seal (Repeat steps 5-7). 8. Once a good alignment and seal are obtained, tighten the clamp screw until it is fingertight. Do not over tighten, plates may crack. 9. For assembling a 16 x 16 cm or 16 x 10 cm CDGE or parallel gel, go to step 16.
Notched Step Comb Gasket Screws Tilt Rod Gasket Holder Fig. 6.5 Comb gasket and holder. Note: Ensure that the comb gasket and it components are free of any gel material. Remove any polymerized gel material in the comb gasket air vents. The soft part of the comb gasket should be snugged within the comb gasket holder. There should be no “wavy” areas on the soft gasket. Use a sturdy, flat spatula to press the soft gasket straight down into the holder. 12.
Gasket Holder Pressure Clamp Pressure Clamp Screw Comb Gasket Air Vent Large Glass Plate Small Glass Plate Fig. 6.6 Pressure clamp assembly. 14. Tighten the comb gasket screws an additional one to one and a quarter turns. If it is tightened more, the glass plate may crack. For a proper seal, check to see that the notches on both the comb gasket and spacers are butted/scrunched against each other. It is important that the gasket is placed properly to prevent leakage while casting.
6.2 Model 475 Gradient Delivery System The Model 475 Gradient Delivery System is used with the D GENE system to construct reproducible linear polyacrylamide gradient gels. Refer to the Model 475 Gradient Delivery System instructions for information on its set-up and use. The Model 475 Gradient Delivery System has a 7–50 ml capacity, making it ideal for the construction of 16 x 16 cm or 7.5 x 10 cm polyacrylamide gradient gels.
6. Prepare the high and low density gel solution by pipeting the desired amounts into two disposable culture tubes (refer to the Model 475 Gradient Delivery System Instructions, Section 4.2). For visually checking the formation of the gradient, add 100 µl of D GENE dye solution per 5 ml high density solution. 7.
6.4 Casting Parallel DGGE Gels 1. Position the gel assembly by standing it upright (Figure 6.8). Note: Place a comb into the sandwich before casting the gel. Placing a comb after casting the gel will disturb the gradient. Fig. 6.8. Pouring a parallel gradient gel. 2. Refer to the Model 475 Gradient Delivery System manual for details on set-up and operation of the gradient former. 3. Insert and fasten all three metal fittings into the Y-fitting.
6. Slide the tubing from the high density syringe over a metal tube fitting on the Y-fitting (about 0.3-0.5 cm of the tip of the tubing). Do the same for the low density syringe. This sequence of inserting the tubing is extremely important to cast the desired gradients. 7. Hold the needle with the beveled edge against the glass plate in the middle of the sandwich to create a uniform flow. 8. Rotate the cam wheel slowly and steadily to deliver the gel solution.
4. Pour or pipette the gel solution into the sandwich until the gel covers the comb teeth. Properly align the comb in the sandwich. Add more gel solution if needed. 5. Allow the gel to polymerize for about 30–45 minutes.
Section 7 Electrophoresis 7.1 Assembling the Upper Buffer Chamber 1. Lay the inner core down flat on a bench. Seat the white U-shaped gasket onto the core with the flat (non-stepped) side down (Figure 7.1). Make sure the U-shaped gasket is clear of any particles, such as residual gel material, that may cause leakage. Note: To help insure a good buffer seal with the gaskets for the D GENE cell, lubricate the entire front of the gaskets (the shaded portion in Figure 7.
Fig. 7.2. Adapting the sandwich assembley to the core. 5. Turn the core to its other side and repeat steps 1–4 to attach the second gel sandwich. Note: When the gel sandwich has been properly installed, the shorter inside glass plate will be forced against the notch in the U-shaped gasket to create a leak proof seal. Always inspect the contact between the gasket and glass plate to make sure the glass plate is butted against the notch in the gasket and is not resting above or below this notch.
Note: The lid can be attached to the buffer chamber in only one orientation, so that the anode and cathode connections cannot be reversed. 4. Remove the D GENE loading lid from the D GENE lid. 5. Load the samples into the wells using a pipet and a sequencing loading tip. Be careful not to not pierce the gel during sample delivery. A multichannel pipet with 8 tips can be used. Note: The loading volume for the single well comb that is used with the perpendicular gels is about 200–400 µl. 6.
Fig. 7.3. Removing the sandwich assembly from the core. 4. Loosen the single screw of each clamp and remove the clamps from the sandwich. With a spatula or an unused spacer, carefully pry off the shorter glass plate. 5. With a razor blade, gently cut the gel along the spacers. This insures that the gel does not tear when the spacers are removed. Remove the spacers and mark one corner of the gel to distinguish between gels. 7.5 Staining and Photographing the Gel 1.
Section 8 Troubleshooting Always confirm that the line voltage is correct for the D GENE system. 8.
Problem Excessive noise Excessive noise during run Cause Solution Worn fan Damaged pump Pump touches cell cover Replace fan Replace pump Move pump by shortening tubing Replace inlet tube assembly circulation Maximum thickness of gel is 0.
8.2 Applications Problem Solution Perpendicular DGGE Only a single band is seen in the “S” curve when at least two bands are expected. 1. Mix normal and mutant DNA prior to the run. 2. Check PCR reaction products for mutant and normal DNA by sequencing or restriction digestion. Hard to visualize heteroduplex and homoduplex DNA bands. 1. Increase DNA loading. Unknown faint bands 1. Impurity or contamination of PCR product. Poor Gradient. “S” curve not fully seen. 1.
Section 9 Maintenance Maintenance of Equipment D GENE system with lid Remove core and clamps from tank. Replace buffer inside tank with distilled water, turn pump on for 1–2 minutes to rinse pump. Remove water from tank. Core, tank, clamps Rinse thoroughly with distilled water after use. Glass plates, spacer, combs Wash with a laboratory detergent (catalog number is 161-0722), then rinse with distilled water. Always inspect the D GENE system and replace any damaged components before use.
Section 10 References For updated references, please request Bio-Rad’s bulletin 1934. 10.1 Applications in Mutation Detection Electrophoresis Application Reference numbers DGGE Genes 16S rRNA gene ....................................................................52 ABO blood group polymorphism........................................36 Adenine phosphoribosyltransferase gene (APRT)..............186 Adenomatous polyposis coli gene (APC) ...........................
Application Reference numbers HPRT gene...........................................................................10, 19, 24, 25, 72, 79, 91, 92, 111, 136, 152, 166, 176, 178, 192, 200 Human acid beta-glucosidase gene .....................................188 Human hypoxanthine guanine phosphoribosyltransferase gene ..........................................112 Human KRAS2 gene ...........................................................60 Human serotonin receptor gene.......................................
Application Reference numbers Breast cancer ........................................................................141 Cancer, general.....................................................................22, 59 Cataracts/retinal detatchment...............................................23 Colorectal adenomas/tumors ...............................................8, 17, 95, 163 Coronary heart disease.........................................................167 Creutzfeldt-Jakob disease ......................
Application Reference numbers Pituitary tumors....................................................................9, 29 Prion disease.........................................................................68 Prostate cancer ....................................................................100 Pseudohypoparathyroidism .................................................81 Retinoblastoma.....................................................................12, 116 Schizophrenia................................
Application Reference numbers Natural population analysis .................................................118 Polymorphism detection ......................................................123, 126 Prenatal diagnosis/carrier testing.........................................14, 49, 56, 101, 128, 138, 158, 172, 194 RNA molecular mutation detection.....................................195 Sardinian population analysis ..............................................128 Sexual orientation .........................
10.2 Mutation Detection Electrophoresis References Denaturing gradient gel electrophoresis (DGGE) 1. Macke, J. P., Hu, N., Hu, S., Bailey, M., King, V. L., Brown, T., Hamer, D. and Nathans, J., Am. J. Hum. Genet., 53 (4), 844–852 (1993). 2. Ripoll, L., Laplanche, J. L., Salzmann, M., Jouvet, A., Planques, B., Dussaucy, M., Chatelain, J., Beaudry, P. and Launay, J. M., Neurology, 43 (10), 1934–1938(1993). 3. Audrezet, MP., Novelli, G., Mercier, B., Sangiuolo, F., Maceratesi, P., Ferec, C.
28. Costes, B., Girodon, E., Ghanem, N., Chassignol, M., Thuong, N. T., Dupret, D. and Goossens, M., Hum. Mol. Genet., 2 (4), 393–397 (1993). 29. Kas, K., Weber, G., Merregaert, J., Michiels, L., Sandelin, K., Skogseid, B., Thompson, N., Nordenskjold, M., Larsson, C. and Friedman, E., Hum. Mol. Genet., 2 (4), 349–353 (1993). 30. Guldberg, P and Guttler, F., Nucleic Acids Res., 21 (9), 2261–2262 (1993). 31. Miric, A., Vechio, J. D. and Levine, M. A., J Clin. Endocrinol. Metab., 76 (6), 1560–1568 (1993). 32.
54. Vidaud, D., Tartary, M., Costa, J. M., Bahnak, B. R., Gispert-Sanchez, S., Fressinaud, E., Gazengel, C., Meyer, D., Goossens, M., Lavergne, J. M., et al., Hum. Genet., 91 (3), 241–244 (1993). 55. Guillermit, H., Jehanne, M., Quere, I., Audrezet, M. P., Mercier, B. and Ferec, C., Hum. Genet., 91 (3), 233–235 (1993). 56. Mercier, B., Lissens, W., Audrezet, M. P., Bonduelle, M., Liebaers, I. and Ferec, C., Hum. Mutat., 2 (1), 16–20 (1993). 57. Lombes, A., Diaz, C., Romero, N. B., Ziegler, F.
81. Lin, C. K., Hakakha, M. J., Nakamoto, J. M., Englund, A. T., Brickman, A. S., Scott, M. L. and Van Dop, C., Biochem. Biophys. Res. Commun., 189 (1), 343–349 (1992). 82. Pellegata, N. S., Losekoot, M., Fodde, R., Pugliese, V., Saccomanno, S., Renault, B., Bernini, L. F. and Ranzani, G. N., Anticancer Res., 12 (5), 1731–1735 (1992). 83. Russ, I. and Medjugorac, I., Nucleic Acids Res., 20 (20), 5491 (1992). 84. Tachdjian, G., Benabdennebi, M., Guidal, C., Sayada, C., Lapoumeroulie, C. and Elion, J., Hum.
107. Zhou, J., Hertz, J. M. and Tryggvason, K., Am. J. Hum. Genet., 50 (6), 1291–1300 (1992). 108. Schwindinger, W. F., Francomano, C. A. and Levine, M. A., Proc. Natl. Acad. Sci. U S A., 89 (11), 5152–5156 (1992). 109. Aly, A. M., Higuchi, M., Kasper, C. K., Kazazian, Jr. H. H., Antonarakis, S. E. and Hoyer, L. W., Proc. Natl. Acad. Sci. U S A., 89 (11), 4933–4937 (1992). 110. Friedman, E., Gordeladze, J. O., Gejman, P. V., Murtagh, Jr., J. J., Gertch, D. S. and Tu, T., Basic Res. Cardiol.
136. Cariello, N. F., Swenberg, J. A., De Bellis, A. and Skopek, T. R., Environ. Mol. Mutagen., 18 (4), 249–254 (1991). 137. DeMarini, D. M. and Fuscoe, J. C., Environ. Mol. Mutagen., 18 (4), 222–223 (1991). 138. Dubel, J. R., Finwick, R. and Hejtmancik, J. F., Am. J. Med. Genet., 41 (1), 39–43 (1991). 139. Weinstein, L. S., Shenker, A., Gejman, P. V., Merino, M. J., Friedman, E. and Spiegel, A. M., N. Engl. J. Med., 325 (24), 1688–1695 (1991). 140. Hovnanian, A., Duquesnoy, P., Amselem, S.
166. Hovig, E., Smith-Sorensen, B., Brogger, A. and Borresen, A. L., Mutat. Res., 262(1), 63–71 (1991). 167. Navajas, M., Laurent, A. M., Moreel, J. F., Ragab, A., Cambou, J. P., Cuny, G., Cambien, F. and Roizes, G., Hum. Genet., 86 (1), 91–93 (1990). 168. Dudley, C. R., Giuffra, L. A., Tippett, P., Kidd, K. K. and Reeders, S. T., Hum. Genet., 86 (1), 79–83 (1990). 169. Bourguin, A., Tung, R., Galili, N. and Sklar, J., Proc. Natl. Acad. Sci. U S A., 87 (21), 8536–8540 (1990). 170. Weinstein, L. S.
197. Borresen, A. L., Hovig, E. and Brogger, A., Mutat. Res., 202 (1), 77–83 (1988). 198. Leider, J. M., Palese, P. and Smith, F. I., J. Virol., 62 (9), 3084–3091 (1988). 199. Lyubchenko, YuL. and Shlyakhtenko, L. S., Nucleic Acids Res., 16 (8), 3269–3281 (1988). 200. Cariello, N. F., Scott, J. K., Kat, A. G., Thilly, W. G. and Keohavong, P., Am. J. Hum. Genet., 42 (5), 726–734 (1988). 201. Myers, R. M., Maniatis, T. and Lerman, L. S., Methods Enzymol., 155, 501–527 (1987). 202. Lerman, L. S.
225. Lothe, R. A., Fossli, T., Danielsen, H. E., Stenwig, A. E., Nesland, J. M., Gallie, B. and Borresen, A. L., J. Natl. Cancer Inst., 84 (14), 1100–1108 (1992). 226. Borresen, A. L., Andersen, T. I., Garber, J., Barbier-Piraux, N., Thorlacius, S., Eyfjord, J., Ottestad, L., Smith-Sorensen, B., Hovig, E. and Malkin, D., et al., Cancer Res., 52 (11), 3234–3236 (1992). 227. Seruca, R., David, L., Holm, R., Nesland, J. M., Fangan, B. M., Castedo, S., Sobrinho-Simoes, M. and Borresen, A. L., Br. J.
Section 11 Systems, Accessories, and Reagents for Mutation Detection Electrophoresis For updated prices in the U.S., please request Bio-Rad’s bulletin 1935. For specifications, please request Bio-Rad’s bulletin 1936.
Catalog Number Product Description 170-9031 Top Gasket, 1.5 mm, D GENE system 170-9032 D GENE Electrophoresis Reagent Kit 170-9033 Replacement Core Gasket, 2 170-9034 MacMelt Software 170-9038 D GENE Control Reagent Kit 170-9039 Casting Stand, D GENE system 170-9040 Clamp Assembly, 10 cm, 1 pair, D GENE system 170-9041 Clamp Assembly, 16 cm, 1 pair, D GENE system 170-9042 D GENE Model 475 Gradient Delivery System 170-9043 Core, D GENE system 170-9044 Comb, 1 well, 0.
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