Cipher DGGE Electrophoresis Systems DGGEK-4001 DGGEK-4801 INSTRUCTION MANUAL
T A B L E O F C O N T E N T S ENGLISH Important User Information. . . . . . . . . . . . . . . . . . . . . . . . 3-4 Section 1: General Information 1.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.2 Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.3 Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SECTION 1 General Information 1.1 Introduction 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.
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.
2.3 Operating Heater/Stirrer/By-Pass Pump (Controller) 2.3.2 Controller Assembly 1) Attach power cord to heater/stirrer and plug into wall outlet. 1. (comes factory-mounted onto DGGE tank) 1. Temperature Sensor 2. Pump Shaft and Impeller 3. Buffer By-Pass Tubing Adapter 4. Over-Temperature Sensor 5. Heater Coils 2.3.1 Front and Rear Panels 10 1 11 9 8 2 3. 4. 17 7 12 3 6 16 1. 2. 3. 4. 5.
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.
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.
DGGEK Unit Set-Up, con’t. 2.4 Setting Buffer Temperature Power Supply Connection 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: To mains (wall outlet) a. Attach the power supply to the safety interlock. b. Attach the safety interlock to a wall (mains) outlet.
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.
3.2 Gel Casting Techniques- continued 3.2 Gel Casting Techniques- continued B. Vertical gradient gel casting using GM-40 gradient maker and gravity flow. C. Vertical gradient gel casting using gradient maker (GM-40) and a minipump. 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.
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. 3.
3.4 Perpendicular Gel Casting 3.5 Perpendicular Gradient Formation 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”.
SECTION 4 Gel Running Preparations 4.2 Buffer Cycling Connections 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-continued. 5.2 Removing the Gels Buffer Cycling Connections – Dual Cassettes 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.
SECTION 6 Recommended Buffers and Reagents SECTION 7 Maximum Well/Comb Volumes Warning: Do not mix buffer in the DGGE tank! Vertical Comb Options, short wells, overall length = 13.1cm Cat. # 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.
SECTION 8 References SECTION 9 Troubleshooting 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 10 Maintenance of Equipment 10.1 SECTION 11 Ordering Information 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.
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; 1211
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