Instruction Manual Denaturing Gradient Gel Electrophoresis System - 700-0175 kuroGel Verti 2020 DGGE - VWR
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Instruction Manual
Denaturing Gradient Gel
Electrophoresis System
700-0175 kuroGel Verti 2020 DGGE
30868/Rev.B/02-13 1DENATURING GRADIENT GEL
ELECTROPHORESIS SYSTEM
kuroGel Verti 2020 DGGE
WARNING
THESE UNITS ARE CAPABLE OF DELIVERING POTENTIALLY LETHAL
VOLTAGE WHEN CONNECTED TO A POWER SUPPLY AND ARE TO
BE OPERATED ONLY BY QUALIFIED TECHNICALLY TRAINED
PERSONNEL.
PLEASE READ THE ENTIRE OPERATOR’S MANUAL THOROUGHLY
BEFORE OPERATING THIS UNIT.
THESE UNITS COMPLY WITH THE STATUTORY EC SAFETY
REQUIREMENTS:
73/23/EEC: LOW VOLTAGE DIRECTIVE: IEC 1010-1:1990 plus
AMENDMENT 1:1992
EN 61010-1:1993/BS EN 61010-1:1993
89/336/EC: THE EMC DIRECTIVE FOR ELECTROMAGNETIC
COMPATIBILITY
VWR INTERNATIONAL DENATURING GRADIENT ELECTROPHORESIS
UNITS ARE DESIGNED TO GIVE LONG SERVICE AND REPRODUCIBLE
RESULTS IN YOUR LABORATORY. A FEW MOMENTS SPENT READING
THESE INSTRUCTIONS WILL ENSURE THAT YOUR EXPECTATIONS ARE
REFLECTED IN THE SUCCESSFUL USE OF THE APPARATUS.
FIRST CHECK THAT THE APPARATUS HAS BEEN RECEIVED COMPLETE
AND UNDAMAGED FOLLOWING SHIPMENT. ANY FAULTS OR LOSSES
MUST BE REPORTED TO VWR INTERNATIONAL IMMEDIATELY. VWR
INTERNATIONAL CANNOT ACCEPT RESPONSIBILITY FOR GOODS
RETURNED WITHOUT PRIOR NOTIFICATION.
REFER TO THE PACKING LIST AND CHECK THAT ALL COMPONENTS
AND ACCESSORIES ARE PRESENT.
PLEASE RETAIN ALL PACKAGING AND MATERIALS
UNTIL THE WARRANTY PERIOD HAS EXPIRED.
30868/Rev.B/02-13 2SPECIFICATIONS:
APPLICATION: For laboratory use only in the electrophoretic separation of proteins and nucleic
acids.
COUNTRY OF ORIGIN: USA
MANUFACTURER: VWR International BVBA
Researchpark Haasrode 2020
Geldenaaksebaan 464
B-3001 Leuven, Belgium
Phone: +32 (0)16 385 011
Fax: +32 (0)16 385 385
E-mail: info@be.vwr.com
Construction:
• Rugged acrylic construction.
• All acrylic joints chemically bonded.
• Doubly insulated cables, rated safe up to 1,000 volts.
• Nickel plated banana plug, corrosion-free and rated safe up to 1,000 volts.
• Recessed power connectors, integral with the safety lid.
• 0.2mm diameter platinum electrodes, 99.99% pure.
• User replaceable platinum electrodes.
• Silicone rubber dovetail seal provides leak-free sealing and are easy to clean and or replace.
• User-friendly clamping system.
• Wide range of accessories.
Environmental Conditions:
• This apparatus is intended for indoor use only.
• This apparatus can be operated safely up to an altitude of 2,000m.
• The normal operating temperature range is between 40C and 650C.
• Maximum relative humidity 80% for temperatures up to 310C decreasing linearly to 50%
relative humidity at 400C.
• The apparatus is rated POLLUTION DEGREE 2 in accordance with IEC 664. POLLUTION
DEGREE 2, states that: “Normally only non-conductive pollution occurs. Occasionally,
however, a temporary conductivity caused by condensation must be expected”.
30868/Rev.B/02-13 3Safety Precautions
• READ the instructions before using the apparatus.
• Always isolate electrophoresis units from their power supply before removing the safety
cover. Isolate the power supply from the mains FIRST then disconnect the leads.
• DO NOT exceed the maximum operating voltage or current
• DO NOT operate the electrophoresis units in metal trays. It is advisable, to always place the
unit on a plastic safety tray during electrophoresis.
• Following the replacement of a platinum electrode have the unit inspected and approved by
your safety officer prior to use.
• DO NOT fill the unit with running buffer above the maximum fill lines.
• DO NOT move the unit when it is running.
• CAUTION: During electrophoresis very low quantities of various gases are produced at the
electrodes. The type of gas produced depends on the composition of the buffer employed.
To disperse these gases make sure that the apparatus is run in a well-ventilated area.
General Care and Maintenance
• To remove the safety lid, push thumbs down on the plastic lugs and lift the lid vertically with
your fingers.
• Before use clean and dry the apparatus with DISTILLED WATER ONLY. IMPORTANT:
Acrylic plastic is NOT resistant to aromatic or halogenated hydrocarbons, ketones, esters,
alcohol’s (over 25%) and acids (over 25%), they will cause “crazing” especially of the UV
transparent plastic and should NOT be used for cleaning. DO NOT use abrasive creams or
scourers. Dry components with clean tissues prior to use.
• Before use, and then on a monthly basis, check the unit for any leaks at the bonded joints.
Place the unit on a sheet of dry tissue and then fill with DISTILLED WATER ONLY to the
maximum fill line. Any leakage will be seen on the tissue paper. If any leakage is seen, DO
NOT ATTEMPT TO REPAIR OR USE THE APPARATUS, but notify VWR International
immediately.
• After each use, wash all components with a suitable water-soluble disinfectant, to reduce the
risk of infection.
• Ensure that the connectors are clean and dry before usage or storage.
• The cooling plate upper surface cannot be cleaned with acetone. Methanol and ethanol can
be used.
At the End of the Run
1. Turn the power supply settings to zero, turn off mains supply and disconnect the power leads.
2. Visualise the run progression or final separation on a UV transilluminator.
3. After each use rinse the apparatus with DISTILLED WATER ONLY.
4. After each use, wash all components with a suitable water-soluble disinfectant, to reduce the
risk of infection.
5. IMPORTANT: Acrylic plastic is NOT resistant to aromatic or halogenated hydrocarbons,
ketones, esters, alcohol’s (over 25%) and acids (over 25%), they will cause “crazing”
especially of the UV transparent plastic and should NOT be used for cleaning. DO NOT use
abrasive creams or scourers. Dry components with clean tissues prior to use.
6. Ensure that the connectors are clean and dry before usage or storage.
Cleaning and Storage
After use, thoroughly rinse all components in deionized water and gently dry the nickel plated
banana plugs with a soft tissue. NEVER USE ORGANIC SOLVENTS.
30868/Rev.B/02-13 4PACKING LIST: No. Items Description Cat. No. 1 kuroGel Verti 2020 DGGE System 700-0175 4 Plain Glass Plates 700-7151 2 Notched Glass Plates 700-7150 4 1.0mm Spacers 700-7152 2 1.0mm Comb, 24 Sample Wells 700-7069 2 Spacer Aligner - 1 100ml Gradient Mixer 700-7102 1 kuroGel Verti 2020 Casting Base 700-0176 1 Silicone Seals for Casting Base, PK/2 700-7080 1 Cables with 4mm Connectors, PK/2 700-7165 30868/Rev.B/02-13 5
Operational
kuroGel Verti 2020 + Heated Tank
Unit Dimensions (W x D x H) 28.5 x 15 x 29cm
Inner Tank Dimensions (W x D x H) 27 x 12.5 x 26cm
Plate Dimensions (W x L x D) 20.5 x 20 x 0.4cm
Standard Spacer Dimensions (W x L x D) 2 x 20 x 0.1cm
Active Gel Dimensions (W x L) 16.5 x 17.5cm
Sample Capacity 2 x 24
Inner Buffer
700ml
Recommended Buffer Volume Chamber
Gel Tank 4200ml
175 - 225V
Voltage
Recommended Running Conditions for (8.75 – 11.25V/cm)
Denaturing/Native PAGE Gel Current 20 - 30mA
Time 2.5 - 3.5h
Temperature Range Thermometer 0 - 100˚C
Power Output of Heating Element 400W
Heating Element Dimensions (L x D) 14 x 1.5cm
Operating Range of PT100 Temperature Sensor -50 to +200˚C
PT100 Temperature Sensor Dimensions (L x D) 14 x 0.3cm
Casting Base Silicone Seal Dimensions (W x L x
1.7 x 22.5 x 0.8cm
H)
Power Output Connectors (diameter) Shrouded, 4 mm
Recommended Power Supplies Consort EV243
HS-UNIT
Unit Dimensions (W x D x H) 9.6 x 14 x 8cm
Recommended Temperature Control Range 50 to 70˚C
Temperature Control Accuracy +/-1˚C
GM100
Unit Dimensions (W x D x H) 11 x 3 x 13.5cm
Internal Dimensions of Mixing and Reservoir
2.5 x 12.5cm
Chambers (Diameter x Height)
Volume of Each Chamber 50ml
Internal Diameter of Outlet Port 2mm
30868/Rev.B/02-13 6USING THE kuroGel Verti 2020 DGGE SYSTEM
The kuroGel Verti 2020 DGGE System is a cost-effective solution for researchers studying mutations and
DNA polymorphisms critical in disease aetiology and genetic diversity. Designed primarily for parallel
denaturing gradient gel electrophoresis (DGGE), where electrophoresis and the denaturing gradient run
in the same direction, the kuroGel Verti 2020 DGGE has a maximum 96-sample throughput compatible
with standard microplates and thermal cycler blocks. A 100ml gradient mixer is also included to pour
gradient gels using our newly designed gel-running module and cam-caster, while a 400W heater,
regulated by an external temperature control unit connected to a heat sensor within the gel tank, allows
the gel temperature to be set to the predetermined melting temperature (Tm) of the PCR®-amplified DNA
polymorphism or mutation of interest.
PT100 Heat Sensor & 400W HS-UNIT Heat sensor control GM-100 – 100ml
Gradient Heating Element Unit – manually adjustable in mixer for 2 x
1mm 10C increments to the desired Tm gradient gels
The protocol in brief
DGGE gels will be poured and run to separate similarly sized PCR products. You will create
gels by combining two solutions containing acrylamide (structural material) and differing
amounts of denaturants (urea and formamide) to form a gradient of denaturant in which
double stranded DNA fragments of differing sequence will be denatured during
electrophoresis. The gel will be stained and visualized to reveal band patterns that can be
used to determine the similarity of sampled microbial communities.
Gel Plate Preparation
1. Clean the plates, spacers and combs in mild laboratory detergent. DO NOT use abrasive
creams or scourers. If a particularly clean finish is required (e.g. for silver-stained gels) glass
plates can be soaked in chromic acid overnight, rinse with water then wipe successively with
ethanol, acetone and ethanol again. NEVER allow organic solvents or chromic acid to come
into contact with the plastic components
2. The notched glass plate can be siliconized in a fume hood with dimethyldichlorosilane if
required to assist in plate separation after the run.
3. Handle clean plates with gloved hands (remove any fingerprints with acetone).
Gel Plate Assembly
1. On a clean level bench, position the two side spacers flush with the edges of the rectangular
glass plate and then overlay the notched plate.
2. The gel plates can be sealed either with tape, or by clamping greased spacers between the
plates with bulldog clips, or by using a casting base, or 2- or 10-gel multicasting units (VWR
product number 700-7046, 700-7056).
30868/Rev.B/02-13 73. To seal with tape, clamp the plates firmly and seal the edges of the gel cassette with gel
sealing tape. The tape should be applied smoothly with no wrinkles. Reinforce the corners
by overlapping extra pieces of tape onto the glass. Grease or fingerprints will prevent a
good seal being formed.
4. If greasing is the preferred method, smear a little silicone grease or Vaseline over the
spacers before assembly and use the long spacer to seal the bottom of the gel and clamp
with bulldog clips. Note that the side spacers will seem too long if this sealing method is
chosen. The side spacers should be cut to size - make sure the cut is a clean right angle.
Casting using the notched gel-running module
Note: If only running one gel, please use 2 plain glass plates (without spacers) on the other side
of the gel running module as needed to prevent short circuit.
1. Loosen the clamping plates on the gel-running module (GRM) by un-tightening the
screws. Place the gel-running module on its side on a level bench surface, and slide the
gel cassettes, comprising the glass plates and spacers, into the gel-running module until
they meet the notched overhangs. The notched glass plates should face the centre of
the gel-running module.
2. Put the gel-running module in an upright position on the bench surface. If using plain
glass plates without bonded spacers, use the spacer aligner to push the spacers so that
they are perfectly aligned with the bottom of the glass plates (there is no need to do this
with plain glass plates with bonded spacers). Tighten the screws, using your other hand
to hold the plates and spacers in position on the bench surface.
3. Once the gel cassettes are tightened into the gel-running module, invert the gel-running
module to check that the plates and spacers are aligned correctly in the gel-running
module. Readjust the plates and spacers if necessary.
4. Place the gel-running module onto the casting base with the silicone seals sitting in the
grooves and the cam-pin levers pointing into the bench surface.
5. Slot the cam pins into position, turning them in a clockwise direction so that the gel-
running module and gel cassettes are drawn onto the silicone seals. The cam pins
should then point upwards at 90° to the bench surface.
6. Check for leaks by adding 2 ml distilled water into each gel cassette. The glass plates
should be firmly embedded within the silicone seals, ensuring that the gel cassettes
remain leak-free. Discard the water and pour the acrylamide gels.
Gel Pouring
1. For reproducibility and uniform polyacrylamide crosslinking, we recommend using
Electran grade materials and degassing gel solutions before use. Acrylamide solutions
should be stored in a cool, dark environment, such as a refrigerator, and allowed to reach
room temperature before pouring. Avoid exposure to heat and sunlight.
2. Polymerization conditions should be adjusted to effect polymerization within about 15
minutes. Test a small volume in a vial before pouring the gel. As a rough guide 100ml of
degassed 6% acrylamide gel will set in about 5 minutes at room temperature when gently
mixed with 450µl of freshly prepared 10% (w/v) ammonium persulfate (APS) plus 200µl
TEMED. The setting time increases to about 10 minutes if the TEMED volume is reduced to
100µl, and to approximately 15 minutes with 75µl. The amount of catalyst may need to be
reduced under warm conditions. Do not pour under direct sunlight.
30868/Rev.B/02-13 83. Gel pouring can be carried out directly in a gel-casting unit or by clamping a taped gel into
the tank unit.
Use the following table to determine the appropriate composition of the denaturing
gradient gel.
Table 1. DGGE gel composition. (Concentrations in bold are variable for different denaturing
concentrations).
4. Make two solutions of 15 ml volume each; a “low” denaturant concentration solution,
and a “high” denaturant concentration solution. For example, if you wish to make a 40–
55% gradient, then you would make a 40% (low) solution and a 55% (high) solution
based on the reagent volumes in Table 1. The gradient mixer will combine these
during the pouring of the gel to create the gradient within the gel matrix.
5. Add APS and TEMED into each solution and swirl gently to mix. These reagents begin the
polymerization of the acrylamide. At this point you will have approximately 15 minutes to
pour the gel.
Gradient Mixer Assembly
1. Set up the gradient mixer by placing a piece of tubing to the outlet port of the mixing
chamber. Pass the tubing through a peristaltic pump. For polyacrylamide slab gels a
flow rate of 5ml per minute is sufficient. Attach the other end of the tubing to the bottom
port of the mutlicaster gel pouring unit, or fixed in position at the bottom of the gel
plate sandwich in the GRM.
2. Place the gradient mixer on a magnetic stirrer and make sure the unit is level. Place a
magnetic stirring bar in the mixing chamber.
3. Close the stopcock valve between the reservoir chamber and the mixing chamber.
4. Prepare the two starting solutions. Degas the acrylamide solutions prior to
use and add the polymerization reagents at the last minute.
5. When pouring the gels from the bottom with a multicaster system, add the high
denaturing solution to the reservoir chamber. Fill the stopcock valve with this solution by,
opening the valve, allowing the tube in it to fill, and then closing the valve. Add the low
denaturing solution to the mixing chamber.
6. Start the magnetic stirrer.
7. Open the outlet port on the mixing chamber and at the same time start the peristaltic
pump. Open the connecting valve between the two chambers.
30868/Rev.B/02-13 98. Run the acrylamide denaturing gel mix slowly down the inside edge of the
gel cassette. Avoid aeration.
9. Allow all of the solution in the mixing chamber to empty before turning off the peristaltic
pump. Note: failure to empty will result in polymerization in the mixing chamber.
10. Clamp the tube at the bottom of the multicasting unit, or remove the tube carefully from
the centrifuge tube.
11. Overlay 10 mls of 1x TAE i
12. Allow the acrylamide solution to polymerize for at least one hour before dismantling the
gel
13. Remove the 1X TAE buffer with a syringe and with the same syringe add approximately 3
ml of “cap” solution (Table 1) to the top of the polymerized gel. Carefully place the comb
at top at a slight angle between the plates. Be sure to avoid trapping any air bubbles as
the comb is lowered into the cap solution. Let polymerise for at least two hours to
overnight.
Running the gel
1. Prepare approximately 5 liters of 1X TAE and fill the outer buffer chamber, put
about 700 ml aside for later use. Preheat the buffer in the DGGE apparatus to
50°C; this will take about 2 hours.
2. When the temperature is about 50°C interrupt heating and place the GRM with
gels in the buffer chamber and the fill inner reservoir with remaining buffer.
3. Flush each well with buffer to remove any unpolymerized acrylamide. Failure to
do this might result in uneven well floors and unresolved bands. Continue heating until
55°C is reached. Do not add the plate assembly to buffer that is too hot (>550C). This
will cause the plates to crack.
4. Flush each well with buffer again and load approximately 40-50 μl of PCR product
containing loading dye to each well. The volume loaded depends on the success of
the PCR and the number of expected products. Soil samples produce many products,
therefore the maximum volume should be loaded. Conversely, when running single
isolate PCR products, a few microliters will suffice. Reset the temperature to 60°C and
run at 20 V for 10 minutes, then run at 200V for 5 hours (1000V·h).
Staining the gel
1. When the electrophoresis is complete, take apart the apparatus and remove the glass
plates from the gel clamps. Carefully separate the plates, leaving the gel exposed.
2. Stain the gel with the desired stain and visualise the bands using a transilluminator.
Further reading
30868/Rev.B/02-13 10Ercolini, D. 2004. PCR-DGGE fingerprinting: Novel strategies for detection of microbes in food. J. Microbiol. Meth. 56:297-314. Muyzer, G. and K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73:127-141. 30868/Rev.B/02-13 11
Chemical Compatibility Table for VWR Electrophoresis Products
Chemical Code Chemical Code Chemical Code
Acetic acid (5%) S Ethyl alcohol (50%) A Naptha S
Acetic acid (Glacial) D Ethyl alcohol (95%) U Nitric acid (10%) S
Acetic Anhydride A Ethylene dichloride D Nitric acid (40%) A
Acetone D Ethylene glycol S Nitric acid U
concentrate
Ammonia S 2-Ethylhexyl S Oleic acid S
Sebacate
Ammonium Chloride S Formaldehyde S Olive oil S
(saturated) (40%)
Ammonium Hydroxide S Gasoline, regular, S Phenol 5% solution U
(10%) leaded
Hydroxide (10%) S Glycerine Heptane S Soap solution S
(commercial grade) (Ivory)
Ammonium Hydroxide S Hexane S Sodium carbonate S
concentrate (2%)
Aniline D Hydrochloric acid S Sodium carbonate S
(10%) (20%)
Benzene D Hydrochloric acid S Sodium chloride S
concentrate (10%)
Butyl Acetate D Hydrofluoric acid U Sodium hydroxide S
(40%) (1%)
Calcium chloride S Hydrogen peroxide S Sodium hydroxide S
(saturated) (3% solution) (10%)
Carbon tetrachloride U Hydrogen peroxide U Sodium hydroxide S
(28% solution) (60%)
Chloroform D Isooctane S Sodium S
hydrochlorite (5%)
Chromic acid (40%) U Isopropyl alcohol A Sulfuric acid (3%) S
(100%)
Citric acid (10%) S Kerosene (no. 2 S Sulfuric acid (30%) S
fuel oil)
Cottonseed oil (edible) S Lacquer thinner D Sulfuric acid U
concentrate
Detergent Solution S Methyl alcohol A Toluene D
(Heavy Duty) (50%)
Diesel oil S Methyl alcohol U Trichloroethylene D
(100%)
Diethyl ether U Methyl Ethyl Ketone U Turpentine S
Dimethyl formamide U Methylene chloride D Water (distilled) S
Dioctyl phthalate A Mineral oil (white) S Xylene D
Ethyl acetate D
S = Safe (No effect, except possibly some staining)
A = Attacked (Slight attack by, or absorption of, the liquid)
( Slight crazing or swelling, but acrylic has retained most of its strength)
U = Unsatisfactory (Softened, swollen, slowly dissolved)
D = Dissolved (In 7 days or less)
30868/Rev.B/02-13 12DISPOSAL This equipment is marked with the crossed out wheeled bin symbol to indicate that it must not be disposed of with unsorted waste. Instead it is your responsibility to dispose of your equipment correctly at lifecycle-end by handing it over to an authorised facility for separate collection and recycling. It is also your responsibility to decontaminate the equipment in case of biological, chemical and/or radiological contamination, so that the persons involved in the disposal and recycling of the equipment are protected from any potential hazard. For more information about where you can drop off your waste equipment, please contact your local dealer from whom the equipment was purchased originally. By doing so, you will help to conserve natural and environmental resources and you will ensure that your equipment is recycled in a manner that protects human health. Thank you 30868/Rev.B/02-13 13
WARRANTY VWR International guarantees that the unit you have received has been thoroughly tested and meets its published specification. This unit (excluding all accessories) is warranted against defective material and workmanship for a period of twenty four (24) months from the date of shipment ex factory. VWR International will repair all defective equipment returned during the warranty period without charge, provided the equipment has been used under normal laboratory conditions and in accordance with the operating limitations and maintenance procedures outlined in this instruction manual and when not having been subject to accident, alteration, misuse or abuse. No liability is accepted for loss or damage arising from the incorrect use of this unit. VWR International’s liability is limited to the repair or replacement of the unit or refund of the purchase price, at VWR International’s option. VWR International is not liable for any consequential damages. VWR International reserves the right to alter the specification of its products without prior notice. This will enable us to implement developments as soon as they arise. VWR International products are for research use only. A return authorisation must be obtained from VWR International before returning any product for warranty repair on a freight-prepaid basis. WARNING DO NOT attempt to remove the outer casing or make repairs to our electrical range of products, should any unit fail. Contact VWR International immediately if the need for repair or servicing should arise. 30868/Rev.B/02-13 14
30868/Rev.B/02-13 15
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