Cipher DGGE Electrophoresis Systems DGGEK-1001 DGGEK-2001 DGGEK-2401 INSTRUCTION MANUAL
T A B L E O F C O N T E N T S Important User Information. . . . . . . . . . . . . . . . . . . . . . . . 3-4 Section 1: General Information 1.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.2 Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6 1.3 Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ENGLISH IMPORTANT USER INFORMATION This Instruction Manual will explain how to use this product safely and effectively. Please read and carefully follow the instruction manual in its entirety. The triangle/exclamation mark symbol alerts the user of the product to important operational, maintenance, and/or warranty requirements. The triangle/lighting bolt symbol alerts the user of the product to potentially hazardous electrical exposure.
FRANÇAIS INFORMATION IMPORTANTE À L’USAGE DES UTILISATEURS ESPAÑOL Le présent manuel d’utilisation explique la manière de se servir efficacement du produit en conditions de sécurité. Il est recommandé de soigneusement lire la totalité du manuel, avec ses consignes et ses instructions. El presente instructivo explica la manera de usar este producto en forma segura y efectiva. Sírvase leerlo en su totalidad y seguir detenidamente las indicaciones que contiene.
SECTION 1 General Information 1.1 Introduction Denaturing Gradient Gel Electrophoresis (DGGE) is a powerful genetic analysis technique that can be used for detecting single base changes and polymorphisms in genomic (1, 2), cloned, and PCR amplified DNA (3,4). Two of the most valuable uses for DGGE in human genetics are in directly detecting single base changes that cause disease and in detecting polymorphisms with DNA probes for genetic-linkage analysis.
Safety Power to the DGGE systems is to be supplied by an external DC voltage power supply that must be ground isolated so that the DC voltage output floats with respect to ground.
Figure 2-1 Buffer Siphon Pump Assembly Heater Stirrer Single Cassetes Vapor Shield Thermometer Comb (2) Glass Plate Assembly(2) Spacers (2 pairs) Buffer recycling manifold Clamps Gelwrap® (2) Mini-Pump (Optional) DGGE Tank GM-40 2.
2.3 Operating Heater/Stirrer/By-Pass Pump (Controller) 1) Attach power cord to heater/stirrer and plug into wall outlet. (comes factory-mounted onto DGGE tank) 2.3.1 Front and Rear Panels 10 1 11 9 8 2 17 7 12 3 6 16 4 5 15 13 14 Front View Rear View 1. Display 11. Pump Speed Switch 2. Preset Temperature Buttons 12. Identification Label 3. Safety Set Reset Button 13. Pump Inlet 14. Reservoir Purge 15.
2.3.2 Controller Assembly 1. Temperature Sensor 2. Pump Shaft and Impeller 3. Buffer By-Pass Tubing Adapter 4. Over-Temperature Sensor 5. Heater Coils 1. 3. 4. 1. 2. 3. 4. 5. 1 Temperature Sensor Pump Shaft and Impeller Pump Outlet Nozzle1 Over-Temperature Sensor Heater Coils 1 2. 3 5. 2 4 5 Rotates on Immersion Circulators 2.3.3 Controller Specifications Temperature Stability ±0.05°C Readout Accuracy ±0.
2.3.4 Controller Operation 2.3.4.a Reservoir Liquid Level Fill the reservoir with the appropriate buffer. The DGGEK Systems are designed to be used with reagent buffers such as 1xTAE or 1xTBE. On Circulating Baths, the liquid level should be sufficient to cover the heating coils, pump, over-temperature sensor, and at least one inch (25mm) of the temperature sensor.
To change from °F to °C, place the Circuit Breaker/Power Switch in the OFF position and then press and hold the P3 Button while turning the power back ON. NOTE: When the temperature display units are changed, the Software High Limit value and all temperature presets revert to the factory-default values. If a calibration value has been entered, the value will be retained. 2.3.4.
2.3.4.h Local Lockout Feature This feature enables the user to lock all controls on the controller. While the feature is activated, the unit will remain running at the current settings. To activate the local lockout feature, press and hold the Select/Set Knob for 10 seconds. Once locked, the screen will read LLo. When locked, the setpoint decimal point will not flash as usual. Press and hold the Select/Set Knob again for 10 seconds to unlock the controls. Once unlocked, the screen will read CAn. 2.3.4.
2.3.6 Controller Calibrations and Maintenance 2.3.6.a Calibration Calibration allows the user to match the Controller’s bath temperature display to an external reference thermometer. Calibration is performed as follows: Set the desired operating fluid temperature set point and allow temperature to stabilize. Press the P2 and P3 simultaneously and release and repeat until the display reads (oCx.x). Press P1 and hold until (Cal) is displayed. This will take about 2 seconds.
DGGEK Unit Set-Up, con’t. 2.4 Setting Buffer Temperature Warning: Do NOT turn on Heater/Stirrer until tank has been filled with buffer! 1. Turn Heater/Stirrer “On” and set temperature to 60°C according to Section 2.3.4.f Setting the Set Point Temperature (page 11). 2.5 Connecting the Power Supply to Safety Interlock 2. Position EPS-300 X Power Supply on a shelf above the tank. Refer to Figure 2-2 to complete the steps below: a. Attach the power supply to the safety interlock. b.
Power Supply Connection To mains (wall outlet) c d To mains (wall outlet) b a Figure 2-2 15 www.cbsscientific.
SECTION 3 Gel Casting Options 3.1 Preparation/Cleaning of Glass Plate for Gel Casting Hand wash both plates with a high quality lab detergent followed by a complete rinsing with dH2O. Air dry or use a lint-free tissue. Spray/wipe the chosen inner surfaces of the plate set with 95% ethanol and dry with lint-free tissue. 3.2 Gel Casting Techniques. A. Gel Wrap™ Gasket Casting Method. B. Vertical gradient gel casting using GM-40 gradient maker and gravity flow. C.
3. Place the gasketed plate on the lab bench with the tubing side up, and extend the bottom of the plate over the edge of the bench, approximately ¾ of an inch. Place the spacers along side the inside edges of the gasket. Be sure the rounded corner end of each spacer is facing the outside bottom of the plate, following the radius of the glass. 4. Place the notched plate on top of the bottom assembly, starting from the bottom edge and gently easing the plate down.
3.2 Gel Casting Techniques- continued B. Vertical gradient gel casting using GM-40 gradient maker and gravity flow. 1. Place the GM-40, gradient maker, on an elevated magnetic stirrer with a small “flea” stir bar in the cylinder (C-2) closest to the outlet. The gradient maker should be fitted with a leur valve, (V-2) and a 20ga needle with attached tubing to deliver acrylamide to the gel plate sandwich as shown in figure (3.5).
3.2 Gel Casting Techniques- continued C. Vertical gradient gel casting using gradient maker (GM-40) and a minipump. 1. Alternatively, you may choose to use a “mini-pump” or other peristaltic pump to cast gels as shown in fig (3.6). If so, secure the gradient maker to a ring stand and connect the outlet tubing to the mini-pump tubing adapter. Connect tubing from mini-pump to a 20ga. needle for affixing between glass plates. Fig. 3-6 19 www.cbsscientific.
3.2 Gel Casting Techniques- continued D. Vertical gradient gel casting using GM-40 gradient maker, Mini-Pump and Multi-Gel Caster. 1. Clean the gel caster with soap and water and the glass plates with alcohol. 2. Place a separating sheet in the gel caster. 3. Place the back plate (non-eared) on the sheet. 4. Place the spacers on each side of the back plate. 5. Place the top plate (eared) on the spacers to form 1st sandwich. Repeat as required. Continue to Section 3.3 Vertical Gradient Formation. Fig.
3.3 Vertical Gradient Formation To determine the range of gradient appropriate for your fragment analysis, please read the enclosed paper by Myers, Sheffield and Cox, especially section 6.1.3 through 6.2.1 (pages 124 to 126). This gives you an excellent overview of the determination of melting behavior of your fragments. 1. The following is a typical protocol for casting a 40%-60% gradient gel. Refer to Section 3.2 Vertical Gradient Gel Casting, figure 3.5, for apparatus assembly.
3.4 Perpendicular Gel Casting NOTE: Prior to casting, mark the notched glass plate to designate which side is the ‘inside’ and ‘outside’. The same side of the notched plate should always sit against the reservoir gasket. 1. For perpendicular gel casting, locate the spacer which has a channel machined into it on one end which will be referred to as the “channel spacer”.
3.5 Perpendicular Gradient Formation To determine the range of perpendicular gradient appropriate for your fragment analysis, please read the enclosed paper by Myers, Sheffield and Cox, as well as the Methods and Enzymology V. 212 paper by Abrams and Stanton. This gives you an excellent overview of the determination of melting behavior of your fragments. 2. The following is a typical protocol for casting a 40%-60% gradient gel. Refer to Section 3.2 Vertical Gradient Gel Casting, figure 3.
SECTION 4 Gel Running Preparations 4.1 Preparation of the Cassettes 1. After polymerization rinse gel plate assembly with D.I. water to remove excess acrylamide or denaturants from plate exterior. 2. Remove comb and quickly transfer gel sandwich to cassette by removing all #2 clamps. LEAVE GEL WRAP IN PLACE. The Gel Wrap acts as a barrier and prevents perpendicular electrical fields from interfering with outside lanes. Reclamp to cassette using #1 clamps/4 per side.
4.2 Buffer Cycling Connections 1. Fill upper reservoir(s) by attaching recirculating tubing to barb fitting on each upper reservoir (fig. 4-2c,d,e). Rinse loading interface of gel with tank buffer to remove non-polymerized acrylamide or excess urea. Adjust final level of lower reservoir buffer by filling the tank until the level of buffer reaches the underside of the upper reservoir. Buffer Cycling Connections – Two Single Cassettes Fig. 4-2c Fig. 4-2d 25 www.cbsscientific.
4.2 Buffer Cycling Connections-continued. Buffer Cycling Connections – Dual Cassettes Fig. 4-2e SECTION 5 Running Conditions 5.1 Running the Gels 1. Load samples at 1:1 with neutral dye. Load 5-10 ug Genomic DNA/well or 1-2 ug cloned (B-globin)/well. Determine concentration by O.D. 260. 2. Attach black power leads to cassettes as shown in Fig. 5-1. 3. Close lid to engage safety interlock. Turn on power supply to 150V (40mA) constant Volts for 5-7 hours. Fig.
5.2 Removing the Gels 3. After the run is completed, turn off power supply, disconnect recirculating tubes and power leads, remove cassettes, remove glass sandwiches from cassettes. 4. Using a wedge plate separator,cat. # WPS-100, pry plates apart and immerse gel/ plate in buffer tray. Stain with Safe Stain or EtBr (.5ug/ml) for 5-10 minutes. Lift floating gel on plate out of tray and rinse with ddH20. Flip over onto saran wrap and peal off glass plate. View bands on transilluminator.
SECTION 6 Recommended Buffers and Reagents Warning: Do not mix buffer in the DGGE tank! 6.1 Stock Solutions Acrylamide Stock Solution 40% Acrylamide/Bis (37.5:1) For 100ml use the following: 38.93 g of acrylamide 1.07 g of Bis-acrylamide Add dH2O (or RO-H2O) to 100ml. Do not autoclave 50x TAE Gel Running Buffer For 1 liter use the following: 242g Tris Base 57.1ml Glacial acetic acid 100ml .5 M EDTA, pH 8.0 Add dH2O (or RO-H2O) to 1 liter Denaturant Stock Solution (7.
SECTION 7 Maximum Well/Comb Volumes Vertical Comb Options, short wells, overall length = 13.1cm Cat. # VGC-0716-177 VGC-0720-177 VGC-0722-177 VGC-0729-177 VGC-0730-177 VGC-1016-177 VGC-1020-177 VGC-1022-177 VGC-1029-177 VGC-1030-177 VGC-1516-177 VGC-1520-177 VGC-1522-177 VGC-1529-177 VGC-1530-177 Tooth depth (mm) # of Teeth Thickness of Teeth Width of Teeth Recommended Max. Sample Vol./Well (µl)* 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 16 20 22 29 30 16 20 22 29 30 16 20 22 29 30 0.75 0.75 0.
SECTION 8 References 1. S.G. Fischer and L.S. Lerman, (1983) PNAS 80:1579. 2. R.M. Myers, T. Maniatis, L.S. Lerman, (1987) Methods in Enzymology, 155: 501-529. 3. R.M. Meyers, V.C. Sheffield, D.R. Cox (1988) “Genome Analysis: A Practical Approach,” Ed. K Davies, IRL Press, Oxford. PP. 95-13 4. V.C. Sheffield, D.R. Cox, L.S. Lerman and R.M. Meyers, (1989) PNAS, 86:232-236 5. Guldberg, P., Henriksen, K.F., and Guttler, F. (1993).
SECTION 9 Troubleshooting 9.1 Unit Will Not Operate (no heat, cooling, or pumping) • Check that the power cord is plugged in to an operating electrical outlet. • Check that the Circuit Breaker/Power Switch is ON. • Check that the Controller Power Switch is ON. • Check that the Safety Set temperature is higher than the control temperature set point. • Turn power off, press the Safety Set Reset button, and restore power. 9.
SECTION 10 Maintenance of Equipment 10.1 Care and Handling The plastic components of the DGGE Systems are fabricated from acrylic and polycarbonate. Electrodes and connectors are made from pure platinum, stainless steel, and chrome plated brass. As with any laboratory instrument, adequate care ensures consistent and reliable performance. After each use, rinse gel cassettes, spacers and combs with de-ionized water. Wipe dry with a soft cloth or paper towel, or allow to air dry.
SECTION 11 Ordering Information CAT. # DESCRIPTION CIPHER DGGE ELECTROPHORESIS SYSTEMS DGGEK-1001 Cipher DGGE Electrophoresis System, 2-place, 1 dual cassette, Specify 110V or 220V. Gel size 17.7cm (w) x 22cm (l).
34 Cipher DGGE Instructions 2/28/14
13R, 712, 7306, 7312, 812, 8006, 8012, 8002, 8112, 8102, 8106, 8202, 8212, 8206, 8306, 9006, 9012, 9002, 912, 9112, 9102, 9106, 9502, 9512, 9506, 9602, 9612, 9606, 9702, 9712, 9706; 18203, 18214, 18211, 18210, 18201, 18206, 18205, 18208, 18212, 18207, 18225, 18226, 18202; 13270-879, 13270-880, 1112A, 1122S, 1127P, 1146D, 1140S, 1147P, 1162A, 1166D, 1160S, 1167P, 1156D, 1150S, 1157P, 1196D, 1190S, 1197P, 1186D, 1180S, 1187P, 1136D, 1130S, 1137P, 1136-1D, 1130-1S, 1137-1P, 1136-2D, 1130-2S, 1137-2P; 12110-00,
CONTACT INFORMATION Online Ordering www.cbsscientific.com Telephone Local and International: 858-755-4959 Toll Free: 800-243-4959 Sales E-mail Address sales@cbssci.com Technical Service E-mail Address technicalservice@cbssci.com Mailing Address P.O.
