Miele DA 196-2 Manuel d'instructions

D GENE™Denaturing GelElectrophoresis SystemInstruction Manualand Applications GuideCatalog Numbers170-9000 through 170-9070For Technical Service Call

Fig. 2.1. An example of DNA melting properties in a denaturing gradient gel. At a low concentra-tion of denaturants the DNA fragment remains double st

Section 3DGE Background Information3.1 IntroductionIn DGGE, DNA is melted by using chemicals denaturants and increased temperature. Asolution of 100%

Fig. 3.1. An example of wild-type and mutant DNA fragments that were denatured and re-annealedto generate four fragments, two heteroduplexes and two h

presence of two or more melting domains. This is caused by the melting of the first domainwhich leads to an almost complete stop in the gel migration.

denaturant gels usually take about 2–6 hours to reach good resolution between mutant andwild-type DNA fragments. An example of parallel DGGE is shown

In constant denaturing gels, only a single denaturing condition is used in the gel. With theconstant denaturing gels, it is possible to run more sampl

Section 4Sample and Reagent Preparation4.1 Sample PreparationIn some cases, adding a high melting domain to the DNA fragment allows one to analyzemuta

50x TAE Buffer (1 L)Tris base 242.0 gAcetic acid, glacial 57.1 ml0.5 M EDTA, pH 8.0 100.0 mlMix and add dH2O to 1 L. Autoclave for 20–30 minutes. Stor

dH2O 6860.0 ml4.3 Gel VolumesThe final gel volumes to use for the three different gel sizes are listed below. Spacer Thickness 16 x 16 cm gel 16 x 10

Section 5Buffer Temperature5.1 Temperature ControllerThe temperature controller maintains the desired buffer temperature in the D GENEsystem (Figure 5

WarrantyThe D GENE lid, tank, casting stand, gradient mixer, and accessories are warranted againstdefects in materials and workmanship for 1 year. If

Section 6Gel Casting6.1 Assembling the Glass Plate SandwichesTo insure proper alignment, make sure all plates and spacers are clean and dry beforeasse

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 alignmentslo

7. Remove the alignment card. Remove the sandwich assembly from the casting stand andcheck that the plates and spacers are flush at the bottom. If th

Fig. 6.5 Comb gasket and holder.Note: Ensure that the comb gasket and it components are free of any gel material. Removeany polymerized gel material i

Fig. 6.6 Pressure clamp assembly.14. Tighten the comb gasket screws an additional one to one and a quarter turns. If it is tight-ened more, the glass

6.2 Model 475 Gradient Delivery SystemThe Model 475 Gradient Delivery System is used with the D GENE system to constructreproducible linear polyacryla

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 D

6.4 Casting Parallel DGGE Gels1. Position the gel assembly by standing it upright (Figure 6.8).Note: Place a comb into the sandwich before casting the

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

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 solut

Table of ContentsPageSection 1 Equipment Overview ...11.1 Safety ...

Section 7Electrophoresis7.1 Assembling the Upper Buffer Chamber1. Lay the inner core down flat on a bench. Seat the white U-shaped gasket onto the co

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:

Note: The lid can be attached to the buffer chamber in only one orientation, so that theanode and cathode connections cannot be reversed.4. Remove the

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. Witha spatul

Section 8TroubleshootingAlways confirm that the line voltage is correct for the D GENE system.8.1 EquipmentProblem Cause SolutionControllerNo display

Problem Cause SolutionExcessive noiseExcessive noise Worn fan Replace fanduring runDamaged pump Replace pumpPump touches cell cover Move pump by shor

8.2 ApplicationsProblem SolutionPerpendicular DGGEOnly a single band is seen 1. Mix normal and mutant in the “S” curve when at DNA prior to the run.

Section 9MaintenanceMaintenance of EquipmentD GENE system with lid Remove core and clamps from tank. Replace buffer inside tank with distilled water,

Section 10ReferencesFor updated references, please request Bio-Rad’s bulletin 1934.10.1 Applications in Mutation Detection ElectrophoresisApplication

Application Reference numbersHPRT gene...10, 19, 24, 25, 72, 79, 91, 92,111, 1

ii

Application Reference numbersBreast cancer...141Cancer, general...

Application Reference numbersPituitary tumors...9, 29Prion disease...

Application Reference numbersNatural population analysis ...118Polymorphism detection...

10.2 Mutation Detection Electrophoresis ReferencesDenaturing gradient gel electrophoresis (DGGE)1. Macke, J. P., Hu, N., Hu, S., Bailey, M., King, V.

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,

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.,

81. Lin, C. K., Hakakha, M. J., Nakamoto, J. M., Englund, A. T., Brickman, A. S., Scott, M. L. and VanDop, C., Biochem. Biophys. Res. Commun., 189 (1

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

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,

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.

Section 1Equipment Overview1.1 Safety Read the manual before using the D GENE system. For technical assistance, contact yourlocal Bio-Rad Office or, i

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)

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), 1100–1

Section 11Systems, Accessories, and Reagents for MutationDetection ElectrophoresisFor updated prices in the U.S., please request Bio-Rad’s bulletin 19

CatalogNumber Product Description170-9031 Top Gasket, 1.5 mm, D GENEsystem170-9032D GENEElectrophoresis Reagent Kit170-9033 Replacement Core Gasket, 2

Life ScienceGroup2000 Alfred Nobel DriveHercules, California 94547Telephone (510) 741-1000Fax: (510) 741-5800www.bio-rad.comAustralia, Bio-Rad Laborat

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

DC Power RequirementExternal DC voltage power supply. This power supply must be ground isolated in such away that the DC voltage output floats with re

the core to provide a greaseless, leak-free seal for the upper buffer. Each sandwich forms oneside of the cathode chamber. If only one gel is to be ru

Section 2Introduction to Technology2.1 Overview of Denaturing Gradient Gel ElectrophoresisThere is an increasing need for practical, efficient, and in