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BIO RAD DCODE Manual

BIO RAD DCODE Manual

Universal mutation detection system 170-9080;170-9104
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DC
™
HE
ODE
U
NIVERSAL
M
UTATION
D
S
ETECTION
YSTEM
Catalog Numbers
170-9080 through 170-9104
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

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Summary of Contents for BIO RAD DCODE

  • Page 1 ™ NIVERSAL UTATION ETECTION YSTEM Catalog Numbers 170-9080 through 170-9104 For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
  • Page 2 © 1996 Bio-Rad Laboratories All Rights Reserved † The DCode system tank is not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene), or acetone. Use of organic solvents voids all warranties. * EN61010-1 is an internationally accepted electrical safety standard for laboratory instruments.

  • Page 3: Table Of Contents

    Table of Contents Section 1 General Safety Information ...............1 Caution Symbols ......................1 Precautions During Set-up ..................1 Precautions During a Run ..................1 Precautions After a Run .....................2 Safety ..........................2 Section 2 Introduction ....................2 Introduction to Mutation Detection Technology ............2 Section 3 Product Description ..................2 Packing List ........................2 System Components and Accessories ...............5...
  • Page 4 Section 6 Single-Stranded Conformational Polymorphism (SSCP).....48 Introduction to SSCP ....................48 Reagent Preparation ....................50 Gel Volumes......................52 Sample Preparation ....................52 Temperature Controller....................52 Cooling the Running Buffer and Chiller Settings ...........53 Assembling the SSCP Gel Sandwich ..............53 Casting SSCP Gels....................55 Section 7 Protein Truncation Test (PTT) ..............56 Introduction to PTT ....................56 Reagent Preparation ....................57 Gel Volumes......................59...

  • Page 5: General Safety Information

    Bio-Rad Office or in the U.S., call Technical Services at 1-800-4BIORAD (1-800-424-6723). DC power to the DCode system is supplied by an external DC voltage power supply. This power supply must be ground isolated so that the DC voltage output floats with respect to ground. All Bio-Rad power supplies meet this important safety requirement.

  • Page 6: Precautions After A Run

    Gradient Gel Electrophoresis (TTGE). Bio-Rad reduced this technique to a simple, reproducible method on the DCode system. TTGE uses a polyacrylamide gel containing a constant concen- tration of urea. During electrophoresis, the temperature is increased gradually and uniformly. The result is a linear temperature gradient over the course of the electrophoresis run. Many labs used combination of methods to maximize mutation detection efficiency.
  • Page 7 The DCode System for DGGE (10 cm and 16 cm systems) Item Quantity Instruction manual Warranty card (please complete and return) Electrophoresis/temperature control module Electrophoresis tank Casting stand with sponges Sandwich core DCode lid stand 16 cm glass plates (16 cm system)
  • Page 8 Glass plates, 20 cm 2 sets Spacers, 0.75 mm 2 sets 20-well combs, 0.75 mm Control reagent kit for SSCP DCode System for Heteroduplex Analysis Item Quantity Instruction manual Warranty card (please complete and return) Electrophoresis/temperature control module Electrophoresis tank...

  • Page 9: System Components And Accessories

    DCode System for Protein Truncation Test Item Quantity Instruction manual Warranty card (please complete and return) Electrophoresis/temperature control module Electrophoresis tank Casting stand with sponges Sandwich core DCode lid stand Sandwich clamps 2 sets Glass plates, 20 cm 2 sets...
  • Page 10 The control module contains the heater, stirrer, pump, and Control Module electrophoresis leads to operate the DCode system (Figure 3.3). Combined with the lower buffer tank, the control module acts to fully enclose the system. The control module should be placed so that the tip of the stirring bar fits inside the support hole of the tank.
  • Page 11 Casting Stand The casting stand holds the gel sandwich upright while casting a gel (Figure 3.5). With the cam levers engaged, the sponge seals the bottom of the gel while the acrylamide polymerizes. Fig. 3.5. Casting Stand. Sandwich Clamps The sandwich clamps consist of a single screw mechanism which makes assembly, alignment, and disassembly of the gel sandwich an effortless task.
  • Page 12 Comb and Spacer Set Different types of combs and spacers are provided with the different DCode systems. Spacer lengths are 10 cm, 16 cm or 20 cm, with a thickness of 0.75 or 1.0 mm. Combs come with 16 or 20 wells and a thickness of 0.75 or 1.0 mm.
  • Page 13 (Figure 3.9). DCode Lid Stand The DCode lid stand provides a stand when the elec- trophoresis/temperature control module (lid) is not in use. The stand must be used to properly support and protect the lid components when the lid is not on the electrophoresis tank.
  • Page 14 Model 475 System Components Description (DGGE System only) Syringe Two pairs each of 10 and 30 ml disposable syringes are provided. The 10 ml syringes are for gel volumes less than 10 ml. The 30 ml syringes are for gel volumes greater than 10 ml.

  • Page 15: Denaturing Gel Electrophoresis (Dgge, Cdge, Ttge)

    Section 4 Denaturing Gel Electrophoresis (DGGE, CDGE, TTGE) 4.1 Introduction to Denaturing Gradient Gel Electrophoresis (DGGE) Denaturing Gradient Gel Electrophoresis (DGGE) is an electrophoretic method to identify single base changes in a segment of DNA. Separation techniques on which DGGE is based were first described by Fischer and Lerman.
  • Page 16 1 kb in length, more than one PCR reaction should be performed. The thermodynamics of the transition of double-stranded to single-stranded DNA have been described by a computer program developed by Lerman. Bio-Rad offers a Macintosh ® computer program, MacMelt ™...

  • Page 17: Reagent Preparation

    The concentration of denaturant to use varies for the sample being analyzed with the DCode system. Typically a broad denaturing gradient range is used, such as 0–100% or 20–70%. The concentration of acrylamide can also vary, depending on the size of the fragment analyzed.
  • Page 18 Polyacrylamide gels are described by reference to two characteristics: 1) The total monomer concentration (%T) 2) The crosslinking monomer concentration (%C) gm acrylamide + gm bis-acrylamide %T = x 100 Total Volume gm bis-acrylamide x 100 %C = gm acrylamide + gm bis-acrylamide 50x TAE Buffer Reagent Amount...

  • Page 19: Gel Volumes

    0.05 g 0.5% Xylene cyanol 0.05 g 0.5% 1x TAE buffer 10.0 ml Store at room temperature. This reagent is supplied in the DCode electrophoresis reagent kit for DGGE, CDGE. 2x Gel Loading Dye Reagent Amount Final Concentration 2% Bromophenol blue 0.25 ml...

  • Page 20: Sample Preparation

    95 °C for 5 minutes, then place at 65 °C for 1 hour, and let slowly cool to room temperature. Temperature Controller The temperature controller maintains the desired buffer temperature in the DCode system (Figure 4.4). The actual and set buffer temperatures are displayed in °C. The set temperature and the temperature ramp rate (RR) can be adjusted by using the raise and lower buttons.

  • Page 21: Preheating The Running Buffer

    Pre-heating the Running Buffer 1. Fill the electrophoresis tank with 7 L of 1x TAE running buffer. Note: It is recommended that the running buffer not be reused. Reusing the running buffer may affect the migration rate and band resolution. 2.
  • Page 22 4. Grasp the gel sandwich firmly. Guide the left and right clamps onto the sandwich so that the long and short plates fit the appropriate notches in the clamp (Figure 4.6). Tighten the screws enough to hold the plates in place. Fig.
  • Page 23 6. Align the plates and spacers by simultaneously pushing inward on both clamps at the locating arrows while, at the same time, pushing down on the spacers with your thumbs. Tighten both clamps just enough to hold the sandwich in place. Pushing inward on both clamps at the locating arrows will insure that the spacers and glass plates are flush against the sides of the clamps (Figure 4.7).
  • Page 24 11. Stand the sandwich assembly upright on a flat surface. Loosen the comb gasket holder screws until the threads can no longer be seen. Mark an arrow on the middle of the screw head using a permanent marker (this will be the marker for adjusting the proper screw tension). With the comb gasket screws and the long plate facing you, slide the comb gasket holder down over the top of the assembled glass sandwich.

  • Page 25: Casting Perpendicular Gradient Gels

    4. From the stocks solutions, pipet out the desired amounts of the high and low density gel solu- tions into two disposable test tubes, Section 4.1. Optional: To visually check the formation of the gradient, add 100 µl of DCode dye solution per 5 ml high density solution.
  • Page 26 7. Place the LO syringe into the gradient delivery system syringe holder (LO density side) by holding the syringe by the plunger and inserting the lever attachment screw into the lever groove. Do not handle the syringe. It will dispense the gel solution out of the syringe. Casting a perpendicular gel is referred to as a bottom filling method, so place the LO syringe on the correct side of the gradient system.

  • Page 27: Assembling The Parallel Gradient Gel Sandwich

    14. Immediately remove the tubing from the sandwich assembly stopcock. Place the tubing into a beaker of water and reverse the cam on the Gradient Delivery System. This will rinse the tubing and Y-fitting. Remove both syringes from the syringe holder on the gradient delivery system.
  • Page 28 4. Grasp the gel sandwich firmly. Guide the left and right clamps onto the sandwich so that the long and short plates fit the appropriate notches in the clamp. Tighten the screws enough to hold the plates in place (Figure 4.11). Fig.

  • Page 29: Casting Parallel Gradient Gels

    5. From the stocks solutions, pipet out the desired amounts of the high and low density gel solutions into two disposable test tubes (refer to the Section 4.1). Optional: To visually check the formation of the gradient, add 100 µl of DCode dye solution per 5 ml high density solution.
  • Page 30 7. Carefully remove air bubbles from the LO syringe by turning the syringe upside down (plunger cap towards the bench) and gently tapping the syringe. Push the gel solution to the end of the tubing. Do not push it out of the tubing as loss of solution will disturb the volume required to cast the desired gel.

  • Page 31: Introduction To Constant Denaturing Gel Electrophoresis (Cdge)

    15. Place the tubing and needle into a beaker of water and reverse the cam on the Gradient Delivery System. This will rinse the tubing and Y-fitting. Remove both syringes from the syringe holder on the gradient delivery system. Detach the syringe tubing from the Y-fitting. Run or push water out through the syringe, tubing, and Y-fitting several times to get rid of any residual gel solution.

  • Page 32: Reagent Preparation

    Both 0% and 100% denaturant should be made as stock solutions. A 100% denaturant is a mixture of 7 M urea and 40% deionized formamide. Reagents for casting and running CDGE gels are included in the DCode electrophoresis reagent kit for DGGE/CDGE, catalog number 170-9032.
  • Page 33 50x TAE Buffer Reagent Amount Final Concentration Tris base 242.0 g Acetic acid, glacial 57.1 ml 0.5 M EDTA, pH 8.0 100.0 ml 50 mM to 1,000.0 ml Mix. Autoclave for 20–30 minutes. Store at room temperature. 0% Denaturing Solution 6% Gel 8% Gel 10% Gel...

  • Page 34: Gel Volumes

    DCode Dye Solution Reagent Amount Final Concentration Bromophenol blue 0.05 g 0.5% Xylene cyanol 0.05 g 0.5% 1x TAE buffer 10.0 ml Store at room temperature. 2x Gel Loading Dye Reagent Amount Final Concentration 2% Bromophenol blue 0.25 ml 0.05% 2% Xylene cyanol 0.25 ml...

  • Page 35: Temperature Controller

    Temperature Controller The temperature controller maintains the desired buffer temperature in the DCode system (Figure 4.16). The actual and set buffer temperatures are displayed in °C. The set temperature and the temperature ramp rate (RR) can be adjusted by using the raise and lower buttons. The °C/RR button is used to scroll between the two parameters.
  • Page 36 Fig. 4.17. Positioning glass plates, spacers, and clamps. 4. Grasp the gel sandwich firmly. Guide the left and right clamps onto the sandwich so that the long and short plates fit the appropriate notches in the clamp (Figure 4.18). Tighten the screws enough to hold the plates in place.

  • Page 37: Casting Cdge Gels

    6. Align the plates and spacers by simultaneously pushing inward on both clamps at the locating arrows while pushing down on the spacers with your thumbs. Tighten both clamps just enough to hold the sandwich in place. Pushing inward on both clamps at the locating arrows will insure that the spacers and glass plates are flush against the sides of the clamps (Figure 4.19).

  • Page 38: Introduction To Temporal Temperature Gradient Gel Electrophoresis (Ttge)

    With no chemical gradient required, rapid, high-throughput screening is possible. The DCode system allows precise control of the temperature ramp rate measured in °C per hour. Control over the temperature range and ramp rate allows optimum denaturing conditions.

  • Page 39: Calculating The Run Parameters

    To determine the temperature range to use with TTGE, a melting profile of the DNA sequence should be generated using a DNA melting software program, such as Bio-Rad’s MacMelt software. As in DGGE, the addition of a 30–40 base pair GC clamp should be added to one of the PCR primers to insure that the region screened is in the lowest melting domain.

  • Page 40: Reagent Preparation

    The concentration of acrylamide to use varies for the sample being analyzed on the DCode system. Therefore, a 40% stock solution containing acrylamide and Bis-acrylamide (Bis) should be made. Reagents for casting and running TTGE gels are included in the DCode elec- trophoresis reagent kit for TTGE, catalog number 170-9171.
  • Page 41 40% Acrylamide/Bis Solutions (1.25x TAE, 6M urea) Reagent 6% Gel 8% Gel 10% Gel 12% Gel 40% Acrylamide/Bis 6.0 ml 8.0 ml 10.0 ml 12.0 ml 50x TAE 1.0 ml** 1.0 ml** 1.0 ml** 1.0 ml** Urea* 14.4 g 14.4 g 14.4 g 14.4 g TEMED...

  • Page 42: Gel Volumes

    The temperature controller maintains the desired buffer temperature and controls the tem- perature ramp rate in the DCode system (Figure 4.23). The actual and set buffer temperatures are displayed in degrees Celsius. The set temperature and the temperature ramp rate (RR) can be adjusted by using the raise and lower buttons.

  • Page 43: Assembling The Ttge Gel Sandwich

    Assembling the TTGE Gel Sandwich For the temporal temperature gradient gel format, a 16 x 16 cm gel sandwich size is recommended. To insure proper alignment, make sure all plates and spacers are clean and dry before assembling. Use caution when assembling the glass plate sandwiches. Wear gloves and eye protection at all times.

  • Page 44: Casting Ttge Gels

    5. Place the sandwich assembly in the alignment slot (the slot without cams) of the casting stand with the short glass plate forward (Figure 4.26). Loosen the sandwich clamps and insert an alignment card to keep the spacers parallel to the clamps. Note: Always use the alignment slot and alignment card to set the spacers in place.

  • Page 45: Heteroduplex Analysis

    Fig. 4.27. Pouring a TTGE gel. 4. Pour or pipette the gel solution into the sandwich until the gel solution covers the wells of the comb. Straighten the comb to the desired well depth. Add more solution if needed. 5. Allow the gel to polymerize for about 60 minutes. After polymerization, remove the comb by pulling it straight up slowly and gently.

  • Page 46: Reagent Preparation

    DCode system. Therefore, a 40% stock solution containing acrylamide and bis-acrylamide (bis) should be made, or a 2x DEM solution. Reagents for casting and running a heteroduplex analysis gel are included in the DCode electrophoresis reagent kit for Heteroduplex Analysis, catalog number 170-9173.
  • Page 47 40% Acrylamide/Bis Solutions (1x TBE) Reagent 6% Gel 8% Gel 10% Gel 12% Gel 40% Acrylamide/Bis 6 ml 8 ml 10 ml 12 ml 10x TBE (see note) 4 ml 4 ml 4 ml 4 ml Urea (optional) see note see note see note see note...

  • Page 48: Gel Volumes

    CSGE Analysis For a different percent crosslinking, use the equation in the heteroduplex analysis reagent preparation to determine the amount of crosslinker to add. The example stock solution below is for an acrylamide/PDA ratio of 99:1. 40% Acrylamide/PDA (99:1) Reagent Amount Acrylamide 198.0 g...

  • Page 49: Sample Preparation

    The temperature controller maintains the desired buffer temperature and controls the temperature ramp rate in the DCode system (Figure 5.1). The actual and set buffer temperatures are displayed in degrees Celsius. The set temperature and the temperature ramp rate (RR) can be adjusted by using the raise and lower buttons.

  • Page 50: Assembling The Heteroduplex Analysis Gel Sandwich

    5.7 Assembly of a Heteroduplex Analysis Gel Sandwich For the heteroduplex gel format, a 20 x 20 cm gel sandwich is recommended. To insure proper alignment, make sure all plates and spacers are clean and dry before assembly. Use caution when assembling the glass plate sandwiches.

  • Page 51: Casting Heteroduplex Analysis Gels

    5. Place the sandwich assembly in the alignment slot (the slot without cams) of the casting stand with the short glass plate forward (Figure 5.4). Loosen the sandwich clamps and insert an alignment card to keep the spacers parallel to the clamps. Note: Always use the alignment slot and alignment card to set the spacers in place.

  • Page 52: Single-Stranded Conformational Polymorphism (Sscp)

    Fig. 5.5. Pouring a heteroduplex analysis gel. 4. Pour or pipette the gel solution into the sandwich until it covers the wells of the comb. Straighten the comb to the desired well depth. Add more solution if needed. 5. Allow the gel to polymerize for about 60 minutes. After polymerization, remove the comb by pulling it straight up slowly and gently.
  • Page 53 The external chiller is set to chill the coolant to approximately -20 °C, and the DCode heater regulates the buffer temperature. An example of an SSCP gel run on the DCode system is shown in Figure 6.1.

  • Page 54: Reagent Preparation

    The concentration and type of acrylamide to use varies for the sample being analyzed on the DCode system; therefore, a 40% stock solution containing acrylamide and bis-acrylamide (bis) should be made. Reagents for casting and running an SSCP gel are included in the DCode electrophoresis reagent kit for SSCP, catalog number 170-9172.
  • Page 55 Using Acrylamide and Bis-acrylamide Stock Solutions Use these calculations to determine the necessary volumes of stock acrylamide and bis-acrylamide solutions to produce gels of any percent and volume. To determine the monomer and crosslinker ratios: (A) is the part of total monomer that is acrylamide gm acrylamide gm acrylamide + gm bis-acrylamide (B) is the part of total monomer that is bis-acrylamide...

  • Page 56: Gel Volumes

    6.5 Temperature Controller The temperature controller maintains the desired buffer temperature and controls the temperature ramp rate in the DCode system (Figure 6.2). The actual and set buffer temperatures are displayed in degrees Celsius. The set temperature and the temperature ramp rate (RR) can be adjusted by using the raise and lower buttons.

  • Page 57: Cooling The Running Buffer And Chiller Settings

    6.6 Cooling the Running Buffer and Chiller Settings 1. To chill the buffer in the DCode system, an external chiller is required. For buffer tem- perature runs between 5–20 °C, the chiller must be able to be set to -20 °C and have a built- in pump to recirculate the coolant (recommended chillers: Haake Model K20 and Lauda Model RMS-6 or equivalent).
  • Page 58 Fig. 6.3. Positioning glass plates, spacers, and clamps. 4. Grasp the gel sandwich firmly. Guide the left and right clamps onto the sandwich so that the long and short plates fit the appropriate notches in the clamp (Figure 6.4). Tighten the screws enough to hold the plates in place.

  • Page 59: Casting Sscp Gels

    Fig. 6.5. Aligning spacers in the sandwich assembly. 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 flush, realign the sandwich and spacers to obtain a good seal (Repeat steps 5–7).

  • Page 60: Protein Truncation Test (Ptt)

    Fig. 6.6. Pouring a SSCP gel. 4. Pour or pipette the gel solution into the sandwich until it covers the wells of the comb. Straighten the comb to the desired well depth. Add more solution if needed. 5. Allow the gel to polymerize for about 60 minutes. After polymerization, remove the comb by pulling it straight up slowly and gently.

  • Page 61: Reagent Preparation

    The concentration and type of acrylamide to use varies for the sample being analyzed on the DCode system, therefore, a 40% stock solution containing acrylamide and bis-acrylamide (Bis) should be made. Reagents for casting and running PTT gels are included in the DCode electrophoresis reagent kit for PTT, catalog number 170-9174.
  • Page 62 The table below provides the percentage acrylamide/bis needed for a particular size range. Gel Percentage Base Pair Separation 7.5% 35–95 kD 15–70 kD 10–40 kD Separating Gel–0.375 M Tris, pH 8.8 Reagent 7.5% 40% Acrylamide/Bis 7.5 ml 12.0 ml (X%) = (A) 1.5M Tris-HCl, pH 8.8 10.0 ml 10.0 ml...

  • Page 63: Gel Volumes

    The temperature controller maintains the desired buffer temperature and controls the temperature ramp rate in the DCode system (Figure 7.1). The actual and set buffer temperatures are displayed in degrees Celsius. The set temperature and the temperature ramp rate (RR) can be adjusted by using the raise and lower buttons.

  • Page 64: Adding The Running Buffer

    7.6 Adding the Running Buffer 1. Remove and place the temperature control module on the DCode lid stand. 2. Add 2 or 7 L of running buffer to the electrophoresis tank. Note: To improve heat dissipation during electrophoresis, 7 L of buffer can be used.
  • Page 65 Fig. 7.3. Attaching the clamps to the glass plate assembly. 5. Place the sandwich assembly in the alignment slot (the slot without cams) of the casting stand with the short glass plate forward (Figure 7.4). Loosen the sandwich clamps and insert an alignment card to keep the spacers parallel to the clamps.

  • Page 66: Casting Ptt Gels

    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 they are not flush, realign the sandwich and spacers for a good seal (repeat steps 5–7). 8.

  • Page 67: Electrophoresis

    Fig. 7.5. Pouring a PTT gel. 10. Pour or pipette the gel solution into the sandwich until it covers the wells of the comb. Straighten the comb to the desired well depth. Add more solution if needed. 11. Allow the gel to polymerize for about 60 minutes. After polymerization, remove the comb by pulling it straight up slowly and gently.
  • Page 68 2. When the running buffer has reached the desired temperature, turn the system off. Disconnect the power cord. 3. Remove and place the temperature control module on the DCode lid stand. Place the core and the attached gel assemblies into the buffer chamber by positioning the red button towards the right hand side and the black button along the left hand side of the system.

  • Page 69: Sample Loading

    Bio-Rad’s Power Pac 300 or 3000. DGGE, CDGE, and TTGE Gels 1. For DGGE and CDGE run the gel at 130 volts. Apply power to the DCode system and begin electrophoresis. As a precaution, always set the voltage, current, and power limits when possible.
  • Page 70 Optional: When using radioactive samples, the pump may be turned off during electrophoresis to reduce radioactive contamination. 3. Apply power to the DCode system and begin electrophoresis. As a precaution, always set the voltage, current, and power limits when possible.

  • Page 71: Removing The Gel

    Note: If different buffers are used between runs it is advisable to rinse the pump with distilled water. Place the DCode module on the DCode stand. Fill a 500 ml beaker with distilled water and place it under the pump inlet tube. Place an empty beaker under the...

  • Page 72: Staining And Photographing The Gel

    Green II (Molecular Probes, Inc.). Stain for about 30 minutes. The gel can also be stained with Radiant ™ Red stain (Bio-Rad catalog number 170-3122). Place the gel into a dish containing 250 ml of running buffer and 1:1,000 dilution Radiant Red stain. Stain for about 30 minutes.

  • Page 73: Troubleshooting Guide

    6. Expose the gel to X-ray film or a phosphor imaging screen. Scan the imaging screen on a storage phosphor imaging system. Section 9 Troubleshooting Always confirm that the line voltage is correct for the DCode system. 9.1 Equipment Problem Cause...
  • Page 74 Dirty inlet fitting Replace fitting or missing O-ring Loose stopcock Tighten stopcock/inlet port screw connection No air vent plug Plug vent after casting gel Damaged or non- Replace with Bio-Rad Bio-Rad glass plates glass plates only...

  • Page 75: Applications

    9.2 Applications Problem Solution Perpendicular DGGE Only a single band is seen Mix normal and mutant DNA samples prior to loading well Difficult to visualize hetero- 1. Increase amount of DNA (1–3 µg). duplex and homoduplex 2. Use SYBR Green I dye agent (Molecular DNA bands Probes, Inc.).
  • Page 76 9.2 Applications (continued) Problem Solution CDGE Normal and mutant Recalculate constant denaturant from a DNA unresolved perpendicular or parallel DGGE gel Air bubbles in gel Clean glass plates Fuzzy DNA bands 1. Clean wells before use. Check for matching comb and spacer thickness. 2.
  • Page 77 Buffer temperature does not 1. Use 50% ethylene glycol as chiller coolant. reach set temperature 2. Insure that DCode temperature controller is set at desired buffer temperature. 3. Check that chiller is set to -20 °C. Air bubbles in gel...
  • Page 78 Fuzzy DNA bands 1. Clean wells before use. Check for matching comb and spacer thickness. 2. Let gel polymerize for at least 60 minutes. Bands did not migrate far 1. Increase run time. enough into gel 2. Decrease acrylamide concentration. 3.

  • Page 79: Specifications

    Section 10 Specifications Construction Tank, core and clamps Tank: molded polycarbonate; Core: molded polysulfone; Clamps: molded glass-filled polycarbonate Urethane or polycarbonate Electrodes 0.010" diameter platinum Electrical leads Polyurethane, copper conductor Casting stand Able to cast two 16 x 20 cm, two 16 x 16 cm, two 16 x 10 cm or one 7.5 x 10 cm gels per setup simultaneously Heater and control Temperature control (PID type) ±...

  • Page 80: Maintenance

    Wash with a laboratory detergent (catalog number 161-0722) then rinse with distilled water. Always inspect the DCode tank, cable, and whole system. Replace any damaged components before use. Damaged parts are to be repaired by Bio-Rad trained personnel with Bio-Rad approved components only.

  • Page 81: Instruments And Reagents For Mutation Detection Electrophoresis

    Section 13 Systems, Accessories, and Reagents for Mutation Detection Electrophoresis For complete ordering information for DCode accessories, electrophoresis reagents, and control kits, request bulletin 2100. Catalog Number Product Description DCode Universal Mutation Detection Systems 170-9080 DCode System for DGGE, 16 cm, 120 V, includes electrophoresis/tem-...
  • Page 82 DCode System for Heteroduplex Analysis, 100 V 170-9098 DCode System for PTT, 120 V, includes electrophoresis/temperature control module, sandwich core, PTT kit for gel casting (2 sets of 20 cm plates, 2 sets of 1 mm spacers, two 20-well 1 mm thick combs)
  • Page 83 40% acrylamide/bis solution(37.5:1), 250 g urea, 225 ml 100% formamide (deionized), 2 x 1 liter 50x TAE buffer, 10 ml of 10 mg/ml EtBr, 1 ml of 2x gel loading dye, 10 ml DCode dye solution, 5 ml TEMED, 10 g ammonium persulfate...
  • Page 85 Latin America, Bio-Rad Latin America, 14100 Palmetto Frontage Road, Suite 101, Miami Lakes, Florida USA 33016 • Phone 305-894-5950 • Fax 305-894-5960 Mexico, Bio-Rad Laboratorios Mexico, Adolfo Prieto No. 1653, Col. De Valle, CP. 03100, Mexico D.F. • Phone 52 5 534 2552 to 54 • Fax 52 5 524 5971 The Netherlands, Bio-Rad Laboratories B.V., Fokkerstraat 10, 3905 KV Veenendaal •...

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