Miele DA 196-2 Instruction manual

D GENE™
Denaturing Gel
Electrophoresis System
Instruction Manual
and Applications Guide
Catalog Numbers
170-9000
through
170-9070
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Warranty
The D GENE lid, tank, casting stand, gradient mixer, and accessories are warranted against
defects in materials and workmanship for 1 year. If any defects occur in the instruments or accessories during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts at
its discretion without charge. The following defects, however, are specifically excluded:
1. Defects caused by improper operation.
2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized
agent.
3. Damage caused by substituting alternative parts.
4. Use of fittings or spare parts supplied by anyone other than Bio-Rad Laboratories.
5. Damage caused by accident or misuse.
6. Damage caused by disaster.
7. Corrosion caused by improper solvent† or sample.
This warranty does not apply to parts listed below:
1. Fuses
2. Glass plates
3. Electrodes
For any inquiry or request for repair service, contact Bio-Rad Laboratories. Inform Bio-Rad of
the model and serial number of your instrument.
IMPORTANT: This Bio-Rad instrument is designed and certified to meet IEC 1010-1* safety
standards. Certified products are safe to use when operated in accordance with the instruction manual. This instrument should not be modified or altered in any way. Alteration of this instrument will:
Void the manufacturer’s warranty
Void the IEC 1010-1 safety certification
Create a potential safety hazard
Bio-Rad Laboratories is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended, or by modifications of the instrument not
performed by Bio-Rad Laboratories or an authorized agent.
† The D GENE 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.
* IEC 1010-1 is an internationally accepted electrical safety standard for laboratory instruments.
Practice of PCR is covered by U.S. patent numbers 4,683,195, 4,683,202, and 4,899,818 issued to Cetus Corporation which is a
subsidiary of Hoffmann-LaRoche Molecular Systems, Inc. Purchase of any of Bio-Rad’s PCR-related products does not convey a
license to use the PCR process covered by these patents; the user of these products to perform PCR must obtain a license.
Copyright 1994 Bio-Rad Laboratories
All Rights Reserved
First Revision
Table of Contents
Page
Section 1
Equipment Overview ..................................................................................1
1.1
1.2
1.3
Safety .........................................................................................................................1
Specifications .............................................................................................................2
Description of Major Components ...........................................................................3
Section 2
Introduction to Technology ........................................................................5
2.1
Overview of Denaturing Gradient Gel Electrophoresis ...........................................5
Section 3
DGE Background Information .................................................................7
3.1
3.2
3.3
3.4
Introduction ...............................................................................................................7
Perpendicular DGGE ................................................................................................8
Parallel DGGE ...........................................................................................................9
CDGE ......................................................................................................................10
Section 4
Sample and Reagent Preparation ...........................................................12
4.1
4.2
4.3
Sample Preparation .................................................................................................12
Reagent Preparation ................................................................................................14
Gel Volumes ............................................................................................................12
Section 5
Buffer Temperature ..................................................................................15
5.1
5.2
Temperature Controller ...........................................................................................15
Pre-heating the Running Buffer ..............................................................................15
Section 6
Gel Casting .................................................................................................16
6.1
6.2
6.3
6.4
6.5
Assembling the Glass Plate Sandwiches ................................................................16
Model 475 Gradient Delivery System .....................................................................21
Casting Perpendicular DGGE Gels .........................................................................21
Casting Parallel DGGE Gels ...................................................................................23
Casting CDGE Gels ................................................................................................24
Section 7
Electrophoresis ..........................................................................................26
7.1
7.2
7.3
7.4
7.5
Assembling the Upper Buffer Chamber .................................................................26
Loading Samples .....................................................................................................27
Running the Gel ......................................................................................................28
Removing the Gel ....................................................................................................28
Staining and Photographing Gel .............................................................................29
Section 8
Troubleshooting Guide .............................................................................30
8.1
8.2
Equipment ...............................................................................................................30
Applications .............................................................................................................32
Section 9
Maintenance ..............................................................................................33
Section 10
References ..................................................................................................34
10.1
10.2
Applications in Mutation Detection Electrophoresis .............................................34
Mutation Detection Electrophoresis References ....................................................39
Section 11
Instruments and Reagents for
Mutation Detection Electrophoresis .......................................................48
i
ii
Section 1
Equipment Overview
1.1 Safety
Read the manual before using the D GENE system. For technical assistance, contact your
local Bio-Rad Office or, in the U.S., call technical services at 1-800-4BIORAD (1-800-4246723).
This instrument is intended for laboratory use only.
This product conforms to the “Class A” standards for electromagnetic emissions intended for laboratory equipment applications. It is possible that emissions from this product may
interfer with some sensitive appliances when placed nearby or in the same circuit as thise
appliances. The user should be aware of this potential and take asppropriate measures to avoid
interference.
DC power to the D GENE 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 of Bio-Rad’s power supplies meet this important safety requirement.
Regardless of which power supply is used, the maximum specified operating parameters for
the system are
Maximum voltage limit
300 VDC
Maximum power limit
50 watts
AC current for controlling temperature to the system, and DC current for electrophoresis,
provided from the external power supply, enter the unit through the lid assembly, which provides a safety interlock. DC current to the cell is broken when the lid is removed. Do not
attempt to circumvent this safety interlock. Always disconnect the AC cord from the
unit and the coiled cord from the DC power supply before removing the lid, or when
working with the cell.
Definition of Symbols
Caution, risk of electric shock
Caution (refer to accompanying documents)
Important precautions during set up
1. Always inspect D GENE system and replace any damaged components before use.
2. Place the D GENE system on a steady, level bench top.
3. Do not use near flammable materials.
4. Do not use buffers which are not compatible with construction material of the system.
5. Always place the lid assembly on the buffer tank with the AC and DC power cords disconnected.
6. Always connect the system to correct the AC and DC power sources.
Important precautions during the run
1. Always add buffer to the “Fill” line on the tank when preheating; always keep the buffer
below “Max” level during electrophoresis.
1
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 top surface of the cover.
Important precautions after the run
1. Always turn off power switches and unplug all cables to DC and AC sources. Allow the
heater tube to cool down (more than 15 seconds) before removing it from the tank.
The ceramic tube may be very hot after shut down. Do not touch the ceramic tube for
several minutes after turning off the power.
2. Do not cool the hot ceramic tube in cool liquids.
3. Store the lid in the aluminum stand for maximum stability. The heater tube may be hot
after use; therefore, support the lid on its own legs, and only on surfaces that can withstand
high temperature.
1.2 Specifications
Construction
Core and clamps
Tank: molded polycarbonate
Core: molded polysulfone
Clamps: molded glass-filled polycarbonate
Gradient former
Cast acrylic and acetyl
Lid
Polycarbonate
Electrodes
0.010” diameter platinum
Electrical leads
Flexible, coiled
Glass plates
16 x 20 cm (16 cm format inner plate)
18.3 x 20 cm (16 cm format outer plate)
10.2 x 20 cm (10 cm format inner plate)
12.4 x 20 cm (10 cm format outer plate)
Gel sizes
16 x 16 cm (max two per run)
16 x 10 cm (max two per run)
7.5 x 10 cm (max four per run)
Spacers available
0.75, 1.0, and 1.5 mm
Combs
16 well comb (compatible with 8 well pipettor), and 1 well comb
(Prep comb for perpendicular gradient gels). Optional: Other
®
combs from PROTEAN II xi system.
Casting stand
Able to cast two 16 x 16 cm, two 16 x 10 cm,
or four 7.5 x 10 cm gels per setup.
Heater and control
Temperature control (PID type) ± 0.5 °C variation within gel area,
± 1.5 °C actual in the range 45 to 65 °C.
Maximum set
temperature
65.5 °C
DC voltage limit
300 V DC
DC wattage limit
50 W
AC Power Requirement
170-9000/9003/9060/9063
AC power input: 120 VAC 47–63 Hz ,5 A slow blow fuse
170-9001/9004/9061/9064
AC power input: 100 VAC 47–63 Hz , 5 A slow blow fuse
170-9002/9005/9062/9065
AC power input: 220–240 VAC 47–63 Hz 2.5 A slow blow fuse
2
DC Power Requirement
External DC voltage power supply. This power supply must be ground isolated in such a
way that the DC voltage output floats with respect to ground.
Maximum voltage limit
300 VDC
Maximum power limit
50 W
Size and Weight
Overall size
Lid and Tank Assembly: 39 cm (L) x 20 cm (W) x 42 cm (H)
Shipping weight
16 Kg
Environmental Requirements
Storage environment
0–70 °C, humidity 0–95% (non-condensing)
Operating environment 0–35 °C, humidity 0–95%.
Regulatory
Meets the requirements of IEC 1010-1 and FCC, Class A.
1.3 Description of Major Components
2
5
3
5
7
1
6
10
8
11
9
4
Fig.1.1. 1. Lower buffer tank. 2. Lid with temperature controller. 3. Core. 4. Comb gasket holder.
5. Sandwich clamps. 6. Casting stand with sponge. 7. Stopcock. 8. Combs and spacer set.
9. Alignment card. 10. Model 475 Gradient Delivery System. 11. Filler spacer.
1. Lower Buffer Tank
The lower buffer tank encloses the unit and provides stability during electrophoresis.
Always keep the buffer level between ‘Fill’ and ‘Maximum’ during a run.
2. Lid with Temperature Controller
Combined with the lower buffer tank, the lid acts to fully enclose the system. The lid
should be placed so that the tip of the stirring bar fits inside the support hole at the tank. The
loading lid should be left on the system at all times unless you are loading samples.
3. Core
The completed gel sandwich attaches to the core so that the outer plate of sandwich forms
the side of the upper buffer chamber. The inner plate is clamped against a rubber gasket on
3
the core to provide a greaseless, leak-free seal for the upper buffer. Each sandwich forms one
side of the cathode chamber. If only one gel is to be run, use a set of glass plates without a spacer to form a buffer dam sandwich. In addition, the core has built-in passage for upper and
lower buffer circulation by the pump.
4. Comb Gasket Holder
The comb gasket holder seals the top edge of the glass sandwich for casting perpendicular gradient gels. Align the comb gasket so that the notched steps on both ends of the soft part
of the comb gasket is against the notches at the top of the two spacers. A proper alignment is
required for a good seal.
5. Sandwich Clamps
The patented sandwich clamps consist of a single screw mechanism which makes assembly, alignment, and disassembly of the gel sandwich an effortless task. The clamps exert an
even pressure over the entire length of the glass plates. Each pair of clamps consists of a left
clamp and a right clamp. Each sandwich can hold a gel up to 1.5 mm thick.
6. Casting Stand with Sponge
The casting stand is separated from the cell so that gels can be prepared for the next run
while others are running.
7. Stopcock and Air Vent Plugs
Stopcocks are used at the inlet ports when casting a gradient perpendicular gel. The air vent
plugs are used in the comb gasket to close the gel sandwich after casting.
8. Prep Comb and Spacer Set
The prep comb works with spacer sets to form a single sample well or dual sample wells
per gel.
9. Alignment Card
The Alignment Card simplifies sandwich assembly by keeping the spacers upright during sandwich alignment.
10. Model 475 Gradient Delivery System
The Model 475 Gradient Delivery System is an innovative cam-driven module for forming accurate, reproducible gradient gels.
11. Filler spacer (optional)
Allows casting of single mini-gel.
12. Pressure Clamp (not shown in photo)
The pressure clamp provides equal pressure to the comb gasket for a good seal.
4
Section 2
Introduction to Technology
2.1 Overview of Denaturing Gradient Gel Electrophoresis
There is an increasing need for practical, efficient, and inexpensive ways to identify
mutations responsible for genetic diseases and cancer development. PCR has solved the
problem of the target limitation. When the precise site of a point mutation is not known, it is
necessary to first determine the region harboring the defect. Six methods are currently available: single-strand conformation polymorphism (SSCP), chemical cleavage (CCM), RNase
mismatch cleavage, reaction of DNA heteroduplexes with a water-soluble carbodiimide (CDI),
direct sequencing, and denaturing gradient gel electrophoresis (DGGE).
RNase cleavage, CDI, CCM, and DGGE use the technique of forming heteroduplex
(annealing between wild-type and mutant DNA or RNA) molecules. These molecules are
characterized by mismatched nucleotides at the sites of alteration. The first three methods are
capable of locating precisely the position of mutation. DGGE depends on electrophoretic
shifts of partially denatured molecules caused by differences in the rate of DNA melting; it can
not be used to detect the exact site of a mutation, but only identify a region containing one or
more mutations. The DGGE method offers the following advantages over the other methods
of mutation detection.
•
The fragment sizes can be > 500 bp, which is larger than allowed by the other
methods and comparable to automated sequencing.
•
The efficiency of detection is approximately 100%.
•
Labeling with radioactivity is unnecessary.
•
DGGE is rapid and easy after the initial set up and is best used for routine screening of
small fragments.
Denaturing gradient gel electrophoresis (DGGE) identifies single base changes in a
segment of DNA. The separation techniques on which DGGE is based were first described by
Fischer and Lerman.212 The separation principle of DGGE involves differences in melting
behavior of DNA fragments which are identical in sequence except for a single base pair.
This melting behavior is detected as a reduction in the mobility of the DNA fragment as it
moves through an acrylamide gel containing denaturing agents, as a consequence of partial
strand separation. In a denaturing gradient polyacrylamide gel, DNA fragments migrate
according to size until they reach the gradient where the molecule begins to denature. When
the DNA denatures, it opens in a domain which is anchored by a higher melting domain or
domains that have not melted. At this point the migration rate is slowed down. When the fragment
completely denatures, then migration again becomes a function of size (see Figure 2.1).
5
Denaturant
0%
100%
Partially melted
“mutant”
Partially melted
“wild type”
Single strands
Electrophoresis
*
Wild Type
*
Mutant
Double strand
Fig. 2.1. An example of DNA melting properties in a denaturing gradient gel. At a low concentration of denaturants the DNA fragment remains double stranded, but as the concentration of denaturants increases the DNA fragment begins to melt. Then, at very high concentrations of denaturants, the
DNA fragment can completely melt, creating two single strands.
The thermodynamics of the transition of double stranded DNA to single stranded DNA
have been described by a computer program developed by Lerman,232 based on the statistical
mechanical principles and algorithms developed by Poland233 and the nearest-neighbor
base-pair doublet parameters introduced by Gotoh and Tagashira.234 There are programs
available which calculate the theoretical DNA melting profile of a given sequence. Bio-Rad
offers Macintosh® computer program, MacMelt™ software, which calculates and graphs
theoretical DNA melting profiles.
Applications of the D GENE system are varied, but its primary purposes are screening for
mutations after in vitro or in vivo mutagenesis, screening for tumor material or acquired
mutations, analysis of candidate genes for possible mutation, analysis of inherited disease,
analysis of new genes, analysis of polymorphism, and marker screening. Other applications
include regulatory protein-nucleic acid complex formation, confirmation of the accuracy of
PCR amplified reaction mixtures, and assay of PCR induced mutations in cloned sequences
without sequencing.
6
Section 3
DGE Background Information
3.1 Introduction
In DGGE, DNA is melted by using chemicals denaturants and increased temperature. A
solution of 100% chemical denaturant consists of 7 M urea and 40% formamide. Denaturing
gels are typically run at temperatures between 50 and 65 °C. The size of the DNA fragments
run on a denaturing gel can be as large as 1 kb in length, but only the lower melting domains
will be available for mutation analysis. For complete analysis of fragments over 1 kb in length,
more than one PCR reaction should be performed.231
When running a denaturing gel, both the mutant and wild-type DNA fragments are run on
the same gel. This way one can detect a mutation by seeing a band shift on the gel. In some
cases, it is possible to detect a mutation by running just homoduplex DNA, for example, if the
mutation is a base change from A to G (G-C pairing has a higher melting temperature than
A-T pairing).201 The method of heteroduplex analysis helps in resolving wild-type and mutated
fragments when it is not possible to detect a mutation by running homoduplex fragments, for
example, if the mutation is a base change from A to T. Heteroduplexes can be formed by
adding the wild-type and mutant template sequence in the same PCR reaction or by adding
separate PCR products together, then denaturing and allowing them to re-anneal. A
heteroduplex has a mismatch in the double strand causing a distortion in its usual shape; this
has a destabilizing effect and causes the DNA strands to separate at a lower concentration of
denaturant (Figure 3.1). The heteroduplex bands always migrate more slowly than the
corresponding homoduplex bands.231
Three types of DGGE gels may be run to detect mutations in DNA. The first gel type is
a perpendicular gradient gel, in which the gradient is perpendicular to the electric field and uses
a broad denaturing gradient range, such as 0–100% or 20–70%.212 From this gel, one can
determine the concentration of denaturants in which the wild-type and mutant fragments can
be separated. The other types of gels are parallel DGGE and constant denaturing gradient gel
electrophoresis (CDGE). In parallel DGGE, the denaturing gradient is parallel to the electric
field, and the range of denaturants is narrowed to allow better separation of fragments.201 In
CDGE there is no gradient. Instead the optimum denaturing concentration which allowed
separation in perpendicular DGGE is used. These three types of gels are discussed in more
detail later in this section.
Starting a DGGE experiment depends on the sequence information available. If the
sequence is known, then PCR can be used to amplify the region of interest. Computer
programs that can calculate the theoretical melting profiles, such as MacMelt software, can
help determine what PCR primers should be used. The theoretical melting profile will also tell
you what part of a gene can be screened for mutations by the system; the primers should be
chosen to flank these regions. A 30-40 base-pair GC clamp (Section 4.1) may be added to
one of the primers to produce a high melting region, so that the rest of the sequence can be
screened for mutations. If the sequence is not known, then a perpendicular DGGE gel with a
large denaturation range (0–100%) should be run to visualize the melting profile of a given
fragment. This will enable one to determine suitable denaturation conditions for subsequent
analysis.
7
Wild Type DNA
Mutant DNA
Denature and reanneal
Homoduplex
DNA
Heteroduplex
DNA
CDGE Gel
wt
mut
wt + mut
Heteroduplexes
Homoduplexes
Fig. 3.1. An example of wild-type and mutant DNA fragments that were denatured and re-annealed
to generate four fragments, two heteroduplexes and two homoduplexes. The melting behavior of
the heteroduplexes is altered so that they melt at a lower denaturant concentration than the homoduplexes, and can be visualized on a denaturing gel, for example a CDGE gel.
3.2 Perpendicular DGGE
Perpendicular DGGE a useful first step in the analysis of any DNA fragment. This technique is used to determine the number of melting domains and the optimum denaturing conditions. If nothing is known about the sequence of interest, a 0–100% gradient gel should be
run. If the sequence information is known, the range of denaturants may be narrowed
(e.g. 20–70%) to improve resolution between the wild-type and mutant fragments.
The perpendicular gel is cast with a preparative comb (1 wide well) and the wild type
and mutant DNA are both loaded into this well. One can run the homoduplex form of the
wild-type and mutant fragments or one can create heteroduplexes. Heteroduplexes can be
formed during PCR or by mixing the mutant and wild-type PCR fragments in a microfuge tube
and denaturing the DNA by heating at 95 °C for 2 minutes, followed by an incubation at
65 °C for 1 hour, and finally incubation at room temperature for 2–20 hours.231 An example
of a perpendicular denaturing gradient gel with homoduplex and heteroduplex fragments is
shown in Figure 3.2.
Perpendicular gels are usually run at 100–165 volts for 2–3 hours or until the xylene
cyanol dye in the loading buffer reaches the bottom of the gel. In some cases, only one melting domain may be seen on the gel, even if the theoretical melting calculations indicate the
8
presence of two or more melting domains. This is caused by the melting of the first domain
which leads to an almost complete stop in the gel migration. This can be resolved by increasing
the run time so that higher melting domains can be seen.
Fig. 3.2. Perpendicular denaturing gradient gel in which the denaturing gradient is perpendicular
to the electrophoresis direction. Mutant and wild-type alleles of exon 6 from the TP53 gene amplified
from primary breast carcinomas and separated by perpendicular DGGE (0-70% denaturant) run at
80 V for 2 hours at 56°C. The first two bands on the left are heteroduplexes and the other two bands are
the homoduplexes.
3.3 Parallel DGGE
For parallel DGGE, the boundary of denaturant concentrations are determined to be above
and below the melting of a given domain as seen in the perpendicular denaturing gel. Examples
for determining the denaturing concentrations are shown in Figure 3.3. Typically, a
difference of 25–30% stock denaturant from top to bottom, which is centered at the melting
point, is used.201 If the melting of two or more domains is seen in the perpendicular gel, then
two different gels with different gradients can be used to maximize separation.
0%
Denaturant
35%
Electrophoresis
50%
100%
65%
Fig. 3.3. Example of a perpendicular denaturing gradient gels used for determining the denaturant
concentration range in a parallel DGGE gel. The DNA fragment melts at a denaturing concentration of
50% and a range of 35–65% denaturants can be used.
In parallel DGGE, the denaturant concentration increases from the top of the gel to the
bottom of the gel. With the parallel gel, it is possible to run more samples under the optimal
conditions. Gel combs are used to form wells in the gel and depending on the number of samples
needed to run, different combs with different number of wells can be used. Parallel gradient
9
denaturant gels usually take about 2–6 hours to reach good resolution between mutant and
wild-type DNA fragments. An example of parallel DGGE is shown in Figure 3.4.
In parallel DGGE, separation is not time dependent for optimal separation. This is due to
the fact that a molecule will migrate with a constant velocity until it reaches the position in the
gel where the denaturant concentration causes the molecule to begin to melt. At this point, the
molecule will migrate more slowly as it moves into higher denaturing conditions.
1
2
3
4
5
6
7
8
-
0%
Denaturants
Electrophoresis
+
40%
Fig. 3.4. Parallel denaturing gradient gel. Time course of wild-type mouse and hamster exon 3
HPRT fragments in a 0-40% parallel DGGE gel. Samples 1, 3, 5, and 7 contains the wild-type mouse
fragment. Samples 2, 4, 6, and 8 contain the wild-type hamster fragment.
3.4 CDGE
For a constant denaturing gel, only one denaturing condition is used to melt the fragment.218
The concentration of denaturant to use for a CDGE is determined at the steepest increase in
gel retardation or the maximum split between wild-type and mutant DNA, as seen in the
perpendicular denaturing gel. An example for determining the denaturing concentrations are
shown in Figure 3.5. If the melting of two or more domains is seen in the perpendicular gel,
two different gels with optimized denaturants are used to maximize separation.
0%
Denaturant
Electrophoresis
48%
100%
Fig. 3.5. Example of perpendicular denaturing gradient gel used for determining the optimum
denaturant concentration used in a constant denaturing gradient gel. The DNA fragment melts at
a denaturing concentration of 48% and this concentration of denaturants can be used.
10
In constant denaturing gels, only a single denaturing condition is used in the gel. With the
constant denaturing gels, it is possible to run more samples under the optimal conditions. Gel
combs are used to form wells in the gel and depending on the number of samples being run,
different combs with different numbers of wells can be used. Constant denaturing gels usually
take about 2–4 hours to reach good resolution between mutant and wild-type DNA fragments.
An example of a constant denaturing gel is shown in Figure 3.6.
In CDGE, separation is time dependent for optimal separation, because wild-type and
mutant fragments immediately begin to partially melt and migrate at a constant rate through
the constant denaturant. Therefore, the running time needed will depend on the resolution
required. A wide separation between wild-type and mutant DNA can be achieved by longer
run times.
1
2
3
4
5
6
7
8
Fig. 3.6. Constant denaturing gel. Amplified mutant and wild-type alleles of exon 8 from the TP53
gene. Separation by CDGE run at 130 V for 2.5 hours on a 10% acrylamide gel in 48% denaturant at
56 °C. Lanes 1 and 5 mutant alleles, lanes 2 and 6 wild-type alleles, lanes 3 and 7 mutant and wild-type
alleles, and lanes 4 and 8 homoduplexes and heteroduplexes of the mutant and wild-type alleles.
11
Section 4
Sample and Reagent Preparation
4.1 Sample Preparation
In some cases, adding a high melting domain to the DNA fragment allows one to analyze
mutations that normally are not seen.210, 211 The addition of a 30–40 base pair GC clamp to the
DNA fragment during PCR creates a high melting domain and will influence the other melting domains.196 As a result, the sequence of interest should be in the first (low) melting domain
and therefore, can be analyzed. A GC clamp is not needed if the sequence of interest resides
in a low melting domain and has one end of the fragment containing a natural high melting
domain. The use of melting profile programs, such as MacMelt software, can show regions
of theoretical high and low melting domains of a known sequence. These programs can help
determine if a GC clamp will allow better detection of mutations.
If GC clamps are not used, this might cause missing of the transition zone and show a
strong single strand band in a perpendicular DGGE gel. In this case, it might be a good idea
to add a GC clamp to one end of the fragment.
As mentioned above, the use of GC clamps helps in the ability to detect mutations. An
alternative to GC clamps is using psoralen derivative PCR primers called ChemiClamp
primers.28 Psoralens are photoreagents that form covalent bonds with pyrimidine bases of
nucleic acids. Psoralen-oligonucleotide conjugates allow crosslinking of DNA fragments at
one end by photoinduction with a UV source. Because ChemiClamps covalently link the two
DNA strands at one end, they should not be used when isolating a DNA fragment which is
going to be sequenced from a gel.
Samples run in the DGGE gel are typically prepared by PCR. The PCR samples can be
loaded onto the gel after PCR without any other manipulations. The size of the DNA fragments
run on DGGE should be in the 100–800 bp range, although DNA fragments as long as 1,000 bp
can also be analyzed.
4.2 Reagent Preparation
The concentration of denaturants varies for samples analyzed in the D GENE system.
The concentration of acrylamide can vary as well, depending on the size of the fragment that
is being analyzed. Both 0% and 100% denaturants should be made as stock solutions. A 100%
denaturant is a mixture of 40% deionized formamide and 7 M urea. The following reagents
are included in the D GENE Electrophoresis Reagent Kit, catalog number 170-9032.
40% Acrylamide/Bis (37.5:1)*
Acrylamide
38.0 g
Bis-acrylamide
2.0 g
Add dH2O to 100 ml. Filter through a Whatman No. 1 and store at 4 °C.
* Polyacrylamide gels are described by reference to two characteristics:
1) The total monomer concentration (%T)
2) The crosslinking monomer concentration (%C)
%T = gm acrylamide + gm Bis-acrylamide x 100
Total Volume
%C =
gm Bis-acrylamide
x 100
gm acrylamide + gm Bis-acrylamide
12
50x TAE Buffer (1 L)
Tris base
242.0 g
Acetic acid, glacial
57.1 ml
0.5 M EDTA, pH 8.0
100.0 ml
Mix and add dH2O to 1 L. Autoclave for 20–30 minutes. Store at room temperature.
0% Denaturing Solution (100 ml)
7.5% Gel
10% Gel
12.0% Gel
40% Acrylamide/Bis (37.5:1)
18.8 ml
25.0 ml
30.0 ml
50x TAE buffer
2.0 ml
2.0 ml
2.0 ml
dH2O
to 100 ml
to 100 ml
to 100 ml
Degas for about 10–15 minutes. Store at 4 °C in a brown bottle for about 1 month.
100% Denaturing Solution (100 ml)
7.5% Gel
10% Gel
12.0% Gel
40% Acrylamide/Bis (37.5:1)
18.8 ml
25.0 ml
30.0 ml
50x TAE buffer
2.0 ml
2.0 ml
2.0 ml
Formamide (deionized)
40.0 ml
40.0 ml
40.0 ml
Urea
42.0 g
42.0 g
42.0 g
dH2O
to 100 ml
to 100 ml
to 100 ml
Degas for about 10–15 minutes. Store at 4 °C in a brown bottle for about 1 month.
For other denaturing solutions, use the volumes in the 100% Denaturing Solution, with the
exception of the formamide and urea. For these reagents, use the volumes indicated below.
Denaturing Solution
Formamide (ml)
Urea (g)
10% 20% 30% 40% 50% 60% 70% 80% 90%
4
8
12
16
20
24
28
32
36
4.2
8.4 12.6 16.8 21
25.2 29.4 33.6 37.8
10% Ammonium Persulfate (1 ml)
Ammonium persulfate
0.1 g
dH2O
1.0 ml
Mix and store at -20 °C for about a month.
D GENE Dye Solution (10 ml)
Bromophenol blue
0.05 g
Xylene cyanol
0.05 g
1x TAE buffer
10.0 ml
Store at room temperature.
1x TAE Running Buffer (7 L)
50x TAE buffer
140.0 ml
13
dH2O
6860.0 ml
4.3 Gel Volumes
The final gel volumes to use for the three different gel sizes are listed below.
Spacer Thickness
0.75 mm
1.00 mm
1.50 mm
16 x 16 cm gel
25 ml
30 ml
45 ml
16 x 10 cm gel
15 ml
20 ml
26 ml
7.5 x 10 cm gel*
8 ml (16 ml)
10 ml (20 ml)
14 ml (24 ml)
* The first volume is for one gel. The volume in parenthesis is for two gels.
Linear Denaturing Gradient Gels
For casting a linear gradient perpendicular and parallel gel, use half the volume of the
low density denaturing solution and half the volume of the high density denaturing solution.
For example, if the total gel volume is 25 ml, use 12.5 ml low density solution and 12.5 ml
high density denaturing solution.
Constant Denaturing Gradient Gels
For casting constant denaturing gradient gels, use the formula below to determine the
volume of 0% and 100% denaturing solutions needed to achieve the desired denaturant
concentration.
1. (% desired denaturant)(total gel volume needed) = ml of 100% denaturant solution
2. (total gel volume needed) - (ml 100% denaturant) = ml of 0% denaturant solution
Example: To cast a 52% constant denaturing gel, using 30 ml total volume for a 16 x 16 cm
gel with a 1.0 mm spacer.
1. (0.52)(30 ml) = 15.6 ml 100% denaturing solution needed
2. (30 ml) - (15.6 ml) = 14.4 ml 0% denaturing solution needed
14
Section 5
Buffer Temperature
5.1 Temperature Controller
The temperature controller maintains the desired buffer temperature in the D GENE
system (Figure 5.1). The actual and set buffer temperatures are displayed in °C. The set
temperature can be adjusted by using the raise and lower buttons. The maximum buffer
temperature that can be set is 65 °C. The heater light will come on when the heater is on.
ACTUAL
HEATER
SET
Fig. 5.1. The Temperature Controller displays the actual and set temperatures.
5.2 Preheating the Running Buffer
1. Pour 1x TAE buffer into the D GENE chamber up to the Fill line. It takes approximately
7 liters of 1x TAE buffer to fill the chamber.
2. Place the D GENE lid onto the chamber. Attach the power cord. Turn the power and
heater switch on. The loading lid should be on the D GENE lid during preheating.
3. Set the temperature 2–4 °C above the desired temperature. During sample loading the
buffer temperature can drop 2–4 °C.
4. It can take about 1 to 1.5 hours for the D GENE system to heat the buffer up to the desired
temperature (56–60 °C). Heating the buffer in a microwave can reduce the time it takes
to get to the desired temperature.
15
Section 6
Gel Casting
6.1 Assembling the Glass Plate Sandwiches
To insure proper alignment, make sure all plates and spacers are clean and dry before
assembly. Use caution when assembling the glass plate sandwiches. Wear gloves and
eye protection.
1. Assemble the gel sandwich on a clean surface. Lay the long rectangular plate down first,
then place the left and right spacers of equal thickness along the short edges of the rectangular plate. For casting perpendicular gradient gels, place the spacers so that the holes
on the spacers are at the top of the plates with the grooved side of the spacer facing the
glass plate and the notched ends facing inwards. For parallel and constant gradient gels,
place the spacers so that the holes in the spacers are at the top of the plate, facing inward
and the grooved side of the spacer facing upward or against the short glass plate.
2. Place a short glass on top of the spacers so that it is flush with one end of the long plate.
3. Locate both the right and left sandwich clamps and loosen the single screw of each clamp
by turning counterclockwise. Place each clamp by the appropriate side of the glass plate
stack with the locating arrows facing up and toward the glass plates (Figure 6.1).
Fig. 6.1. Positioning glass plates, spacers, and clamps.
4. Grasp the whole glass plate 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 6.2).
16
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 alignment
slot is the slot without cams) with the short glass plate forward (Figure 6.3). Loosen the
sandwich clamps and insert a D GENE alignment card to keep the spacers upright.
Note: Always use the alignment slot and alignment card to set the spacers in place. Failure
to use this slot for alignment can result in gel leakage while casting or buffer leakage during
the run.
6. Flush 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 a thumb; tighten
both clamps just enugh 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 6.3).
Fig. 6.3. Aligning spacers in the sandwich assembly.
17
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 flushed, realign the sandwich and spacers to obtain a good seal (Repeat steps 5-7).
8. Once a good alignment and seal are obtained, tighten the clamp screw until it is fingertight. Do not over tighten, plates may crack.
9. For assembling a 16 x 16 cm or 16 x 10 cm CDGE or parallel gel, go to step 16. If you
are assembling a 16 x 16 cm or 7.5 x 10 cm perpendicular gradient gel, go to step 10.
10. Place the proper comb in the sandwich and set it against the special notches provided on
the spacers. For the 7.5 x 10 cm perpendicular gel, insert the middle spacer on the center
of the sandwich until it touches the middle notch on the comb and straighten the spacer.
The bottom of the middle spacer should also be flushed against the glass plates without
any snags (Figure 6.4).
Note: If casting a single 7.5 x 10 cm gel, use the filler spacer, which fills the glass plate
on the side that is not being used.
Fig. 6.4. Positioning the middle spacer in the sandwich assembly.
11. Set the sandwich standing upright on a flat surface. Loosen the comb gasket screws
(located on the back, Figure 6.5). Mark a straight line on the middle of the screw head
using a permanent marker (this will be the marker for the turns needed for the screws).
Carefully place the comb gasket on top of the comb with the screws facing towards the
large plate. Position the comb gasket so that the notched steps at the top of both ends of
the soft part of the comb gasket is against the notches at the top of the spacers. Twist the
screws until it makes contact with the glass plate. Twist the screws an additional 1⁄4 turn.
18
Notched Step
Comb Gasket Screws
Tilt Rod
Gasket Holder
Fig. 6.5 Comb gasket and holder.
Note: Ensure that the comb gasket and it components are free of any gel material. Remove
any polymerized gel material in the comb gasket air vents. The soft part of the comb
gasket should be snugged within the comb gasket holder. There should be no “wavy”
areas on the soft gasket. Use a sturdy, flat spatula to press the soft gasket straight down
into the holder.
12. Loosen the Pressure Clamp screw to be used for the appropriate gel sandwich
size (Figure 6.6). Mark a straight line on the middle of the screw head using a permanent
marker (this will be the marker for the turns needed for the screws). Lay the Pressure
Clamp on a flat surface with the screw upwards.
13. Grasp the gel sandwich with the short glass plate facing up. Do not touch the comb gasket.
Lay the sandwich on top of the Pressure Clamp with the short plate facing upward and the
middle spacer aligned with the Pressure Clamp. The base of the large plate should be
against the “notched” portion of the Pressure Clamp and the Pressure Clamp screw against
the top of the comb gasket and above and between the two air vent plugs/holes. Ensure that
the pressure clamp is positioned in the middle of the comb gasket. This provides equal
force onto the comb gasket for a good seal. Twist the Pressure Clamp screw until it makes
contact with the comb gasket. Tighten the Pressure Clamp screw one and a half turns.
19
Gasket Holder
Pressure Clamp
Pressure
Clamp
Screw
Comb Gasket
Air Vent
Large Glass Plate
Small Glass
Plate
Fig. 6.6 Pressure clamp assembly.
14. Tighten the comb gasket screws an additional one to one and a quarter turns. If it is tightened more, the glass plate may crack. For a proper seal, check to see that the notches on
both the comb gasket and spacers are butted/scrunched against each other. It is important that the gasket is placed properly to prevent leakage while casting. Remove the
Pressure Clamp.
15. Twist a stopcock into each of the injection ports until it is finger-tight. A loose stopcock
may cause leakage during gel casting. Screw the injection port into the holes located on
the sandwich clamps. Do not tighten with the stopcock, it may loosen. Do not overtighten the injection port, it will damage the O-ring and cause a leakage. A snug fit is all that
is needed to place the injection port against the glass plate/spacer.
a. For the 7.5 x 10 cm dual gel sandwich, only one half of the sandwich is cast at a time.
Open the stopcock on the gel sandwich and be sure the air port is unplugged. Be sure
that the ports on the half of the sandwich that is not being cast are closed. This helps
to lessen leakage.
b. For assembling a 16 x 16 cm perpendicular gradient gel sandwich, plug or tape the two
middle holes on the comb gasket to prevent leakage upon casting.
16. The camshafts on the casting stand should have the handles pointing up and pulled out.
Place the gray sponge into the front casting slot. Place the sandwich on the sponge with
the shorter plate facing forward. When the sandwich is placed correctly, press down on
the sandwich clamp screw and turn the handles of the camshaft pointing downwards so
that the cams lock the sandwich in place.
20
6.2 Model 475 Gradient Delivery System
The Model 475 Gradient Delivery System is used with the D GENE system to construct
reproducible linear polyacrylamide gradient gels. Refer to the Model 475 Gradient Delivery
System instructions for information on its set-up and use. The Model 475 Gradient Delivery
System has a 7–50 ml capacity, making it ideal for the construction of 16 x 16 cm or 7.5 x 10 cm
polyacrylamide gradient gels. The mixing of the high and low density solutons occurs in the
Y-fitting and a single outlet leads to the gel sandwich. The gradient may be cast from the top
for parallel gels or from the side for perpendicular gels.
6.3 Casting Perpendicular DGGE Gels
1. Position the gel assembly tilted to about a 20° angle by adjusting the rod on the comb
gasket. This is to insure that any air bubbles that form while casting the gel escapes to the
air vent (Figure 6.7).
Fig. 6.7. Pouring a perpendicular gradient gel using the Model 475 Gradient Delivery System.
2. Refer to the Model 475 Gradient Delivery System manual for details on set-up and
operation of the gradient former.
3. Insert and fasten all three metal fittings into the Y-fitting. Connect the free end of the
9 cm Tygon tubing to one of the metal fittings in the Y-fitting and connect to the other end
a luer coupling. Connect each of the luer fittings on the two long pieces of tubing, then
connect the luer fitted ends to same size syringes. Do not connect the long Tygon tubings
to the Y-fitting.
4. Label one of the syringes LO (for the low density solution) and one HI (for the high density solution). Attach a plunger cap onto each syringe plunger “head.” Tighten enough to
hold the plunger in place. Slide each syringe into a syringe sleeve. Twist and position the
sleeve to the middle of the syringe with the volume gradations visible. Make sure that
the lever attachment screw is in the same plane as the flat or back side of the sleeve. This
is extremely important for proper attachment of the syringe to the lever.
Note: Ensure that the tubing pieces are free of any gel material by simply pushing water through
the tubing with the syringe. The tubings should be free of any materials before casting.
5. Rotate the cam wheel counterclockwise to the start position. At the start point, the lever is in
the vertical position. Adjust the volume adjustment screw to the desired setting. Refer to the
Model 475 Gradient Delivery System instructions, Section 4.1, for the correct setting to use.
21
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 Delivery System Instructions,
Section 4.2). For visually checking the formation of the gradient, add 100 µl of D GENE
dye solution per 5 ml high density solution.
7. Add 1⁄100 of the total gel volume of 10% ammonium persulfate and 1⁄1000 of the total gel
volume of TEMED to the tubes containing the gel solutions (these ratios allow about
3–5 minutes to finish casting the gel before polymerization). Cap and mix by inverting
several times. With the syringe connected to the tubing, withdraw all of the high density
solution using the high density solution syringe. (Excess air is not a problem at this time).
Do the same for the low density solution syringe.
Note: Acrylamide is a very hazardous substance. Use caution: wear gloves and eye
protection. Avoid skin contact.
8. Carefully remove any air bubbles by tapping the syringe. Completely fill the tubing with
gel solution. Do not displace the gel solution, loss of gel solution will result in an incompletely cast gel.
Note: The volume of gel solution should be greater (about 1 ml total volume for both
syringe volumes) than the volume set on the volume adjustment screw (See Section 4.1).
For example, if the volume adjustment screw is set at 4.5, the volume of each gel solution
should be ≥ 5 ml in the syringe in order to have enough gel solution for casting the gel.
The excess gel solution will not affect formation of the desired gradient.
9. Place the high density syringe into the syringe holder (high density side for bottom filling).
Make sure to place the syringe in the correct syringe holder, i.e. the placement of the low
and high density syringes depends on whether the gel is being poured from the top or
bottom. Note: Perpendicular gradient gels are bottom filled. Check for proper high
and low syringe position on the gradient delivery system. Hold the syringe
by the plunger and insert the lever attachment screw into the lever groove. Try not to handle the syringe, it may dispense the gel solution out of the syringe. Carefully position the
lever attachment screw by placing it in the groove and sliding the syringe back towards
the cam side. Tighten the syringe holder screw against the syringe sleeve. Do the same for
the low density solution (low density side for bottom filling).
10. Slide the tubing from the high density syringe over a metal tubing fitting on the Y-fitting
(about 0.3-0.5 cm of the tip of the tubing). Do the same for the low density syringe. This
sequence of inserting the tubings is extremely important to cast the desired gradients.
11. Connect the Y-fitting to the gel sandwich stopcock fitting. Make sure the stopcock on
the gel sandwich is open and the air vent port on the comb gasket is unplugged.
12. Rotate the cam wheel slowly and steadily to deliver the gel solution. It is important to cast
the gel solution at a steady pace to avoid mixing of the gradients within the gel sandwich.
13. Close the air vent port and stopcock on the gel sandwich when the cam wheel has reached
the stop position. Carefully level the gel sandwich by adjusting the gasket tilt rod. Make
sure that you loosen the gasket tilt rod screw and not the sandwich clamp screw.
14. Remove the tubing from the gel sandwich stopcock. Remove both syringes from the
syringe holder on the gradient delivery system. Detach the syringe tubings from the metal
tubings on the Y-fitting. Run or push water out through the tubings and Y-fitting. It is
extremely important that this is done quickly after casting to avoid any gel polymerization (See Gradient Delivery System manual for information on cleaning).
15. Let the gel polymerize for about 30-45 minutes. Remove the gasket tilt rod and place it
on the second gel side of the comb gasket. Repeat steps 6 through 15.
22
6.4 Casting Parallel DGGE Gels
1. Position the gel assembly by standing it upright (Figure 6.8).
Note: Place a comb into the sandwich before casting the gel. Placing a comb after casting the gel will disturb the gradient.
Fig. 6.8. Pouring a parallel gradient gel.
2. Refer to the Model 475 Gradient Delivery System manual for details on set-up and
operation of the gradient former.
3. Insert and fasten all three metal fittings into the Y-fitting. Connect the free end of the 9 cm
Tygon tubing to one of the metal fittings in the Y-fitting and connect to the other end a
luer coupling. Connect each of the luer fittings on the two long pieces of tubing, then
connect the luer fitted ends to same size syringes. Do not connect the long Tygon tubings
to the Y-fitting.
Note: Ensure that the tubings are free of any gel material by simply pushing water through
the tubing with the syringe. The tubing should be free of any material before casting.
4. Repeat steps 4 through 8 in Section 6.3.
5. Place the high density syringe into the syringe holder (high density side for top filling).
Make sure to place the syringe in the correct syringe holder, i.e., the placement of the
low and high density syringes depends on whether the gel is being poured from the top
or bottom. Note: Parallel gradient gels are top filled. Check for proper high and low
syringe position on the gradient delivery system. Hold the syringe by the plunger and
insert the lever attachment screw into the lever groove. Try not to handle the syringe, it
may dispense the gel solution out of the syringe. Carefully position the lever attachment
screw by placing it in the groove and sliding the syringe back towards the cam side.
Tighten the syringe holder screw against the syirnge sleeve. Do the same for the low density solution (low density side for top filling).
23
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 for the low density syringe. This
sequence of inserting the tubing is extremely important to cast the desired gradients.
7. Hold the needle with the beveled edge against the glass plate in the middle of the
sandwich to create a uniform flow.
8. Rotate the cam wheel slowly and steadily to deliver the gel solution. It is important to cast
the gel solutions at a steady pace to avoid mixing of the gradients within the gel sandwich.
9. Remove the tubing from the gel sandwich stopcock. Remove both syringes from the
syringe holder on the gradient delivery system. Detach the syringe tubings from the metal
tube fitting on the Y-fitting. Run or push water out through the tubings and Y-fitting. It is
extremely important that this is done quickly after casting to avoid any gel polymerization
(See Gradient Delivery System manual for information of cleaning).
10. Let the gel polymerize for about 30–45 minutes.
6.5 Casting CDGE Gels
A constant denaturant gel contains the same chemicals as both the perpendicular and parallel gradient gels. The only difference is CDGE has a uniform concentration of denaturant.
1. Assemble a gel sandwich as in Section 6.1 steps 1–12. The gel sandwich is cast in an
upright position.
2. Into a 50 ml tube, add the required amounts of 0% and 100% gel solutions needed to get
the desired percent of denaturant (see Section 4). Add the 1⁄100 the total gel volume of
ammonium persulfate and 1⁄1000 the total gel volume of TEMED. Cap the tubes and mix.
3. Place a comb in the gel sandwich and tilt it so that the teeth are at a slight (~10°) angle.
This will prevent air from being trapped under the comb teeth while pouring the gel
solutions (Figure 6.9).
Fig. 6.9. Pouring a CDGE gel.
24
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 solution if needed.
5. Allow the gel to polymerize for about 30–45 minutes.
25
Section 7
Electrophoresis
7.1 Assembling the Upper Buffer Chamber
1. Lay the inner core down flat on a bench. Seat the white U-shaped gasket onto the core with
the flat (non-stepped) side down (Figure 7.1). Make sure the U-shaped gasket is clear of
any particles, such as residual gel material, that may cause leakage.
Note: To help insure a good buffer seal with the gaskets for the D GENE cell, lubricate
the entire front of the gaskets (the shaded portion in Figure 7.1) with water or upper buffer
prior to attaching the gel sandwich to the central cooling core. This will allow the glass
plate sandwich to slide onto the gasket properly.
Fig. 7.1. Core gasket.
2. After the gel has polymerized, release the gel sandwich from the casting stand by turning
the camshafts 180°, to the up position and pulling them outward. Remove the gel sandwich. Remove any stopcocks and comb gaskets. Remove the comb.
3. With the short glass plate facing the core, position the gel sandwich so that the grooves
in the upper portion of the clamps are fitted onto the locating pins on the core. The gel
sandwich should be positioned at an angle of ≤ 20° with the core. Keeping this angle to
a minimum will prevent distortion of the gasket while the sandwich slides into place.
4. With your fingers below the latch on the core and your thumbs resting on the sandwich
clamps, gently push the gel sandwich onto the core with one simple motion. You should
be able to hear a click. The upper edge of the short inner glass plate should be butted
against the notches of the U-shaped gasket and the tabs of each clamp should be held
securely against the latch assemblies on both sides of the core (Figure 7.2).
26
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: When the gel sandwich has been properly installed, the shorter inside glass plate will
be forced against the notch in the U-shaped gasket to create a leak proof seal. Always inspect
the contact between the gasket and glass plate to make sure the glass plate is butted against
the notch in the gasket and is not resting above or below this notch. Improper installation of
the gel sandwich can result in buffer leakage during the run. As a standard procedure, stand
the core and the attached gel sandwiches, pour buffer into the upper buffer chamber, and
check for leaks prior to a run. Pour enough buffer to immerse the cathode wires connected
in the core. If no leaks are present, it is safe to run the gel.
6. If only one gel is to be run, assemble the other set of glass plates, without the spacers.
Place the short glass plate on top of the long glass plate. Guide the left and right clamps
onto the sandwich so that the plates fit appropriate notches in the clamp. Tighten the
screws enough to hold the plates in place. No further alignment is necessary. Attach it to
the other side of the core to form an upper chamber dam.
Note: Failure to slide the dam up completely to the top of the clamp will result in buffer
leaking from the upper chamber.
7.2 Loading Samples
1. When the running buffer has reached the correct temperature in the D GENE chamber, the
samples are ready to be loaded onto the gel. Turn the D GENE system off. Disconnect the
power cord. Place the core and the attached gel assemblies into the lower buffer chamber.
The core goes in the chamber in only one direction. Add approximately 350 ml of
1x TAE buffer to the upper buffer chamber.
Note: If you want to prerun to equilibrate the system, place the lid on the D GENE
system, attach cords, and adjust the voltage setting on the power supply.
2. Wash the wells using a 19 gauge needle and syringe with the running buffer to remove any
unpolymerized gel material in the wells before loading samples.
3. Place the D GENE lid back onto the buffer chamber. Turn the power and heater on. Adjust
the temperature setting to the desired temperature (see Section 5.1).
27
Note: The lid can be attached to the buffer chamber in only one orientation, so that the
anode and cathode connections cannot be reversed.
4. Remove the D GENE loading lid from the D GENE lid.
5. Load the samples into the wells using a pipet and a sequencing loading tip. Be careful not
to not pierce the gel during sample delivery. A multichannel pipet with 8 tips can be used.
Note: The loading volume for the single well comb that is used with the perpendicular gels
is about 200–400 µl.
6. Place the D GENE loading lid back onto the D GENE lid. Turn the pump on. Adjust to
the correct temperature.
7.3 Running the Gel
1. Attach the electrical leads to a suitable DC power supply with the proper polarity
(this connection could accidentally be reversed).
2. Apply power to the D GENE system and begin electrophoresis. You can also run the
samples into the gel before turning on the pump. As a safety precaution always set
voltage, current, and power limits when possible.
7.4 Removing the Gel
1. After electrophoresis is complete, turn the power supply and D GENE system (heater,
pump, and main power) off. Disconnect the power cord and electrical leads. Let the heater
cool for about 15 seconds in the buffer.
2. Remove the D GENE electrophoresis system and place it on the holder. CAUTION: The
heater is still hot. Do not touch. Carefully pull the core out of the lower buffer chamber.
Pour off the upper buffer into the lower buffer chamber by tilting the core into the chamber.
3. Lay the core and gel sandwiches on a padded surface.
a. For 16 x 10 cm and 7.5 x 10 cm gels, remove the sandwich assembly with your index
fingers on the sandwich clamps and your thumbs resting on the latches on the core.
Gently remove the assembly by pushing the latch away from the gel sandwich. Pry the
sandwich upward and pull (Figure 7.3).
b. For 16 x 16 cm gels, remove the sandwich assembly with your index fingers below the
sandwich clamps and your thumbs resting on the latches on the core. Gently remove
the assembly by pulling up toward you (in a manner opposite to the way it was
attached). Pull the sandwich assembly off the locating pins on the top of the core.
28
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. With
a spatula or an unused spacer, carefully pry off the shorter glass plate.
5. With a razor blade, gently cut the gel along the spacers. This insures that the gel does not
tear when the spacers are removed. Remove the spacers and mark one corner of the gel
to distinguish between gels.
7.5 Staining and Photographing the Gel
1. Remove the gel from the glass plate by gently grasping two corners and lifting off. Place
the gel into a dish containing 250 ml of 1x TAE buffer and 100 µl of 1 mg/ml ethidium
bromide. Stain for about 1–2 minutes. Other dyes such as SYBR-Green I (Molecular
Probes) can be used.
2. After staining, carefully transfer the gel into a dish containing 250 ml of 1x TAE buffer.
Destain for 5-20 minutes.
3. Place the gel on a UV transilluminator and photograph gel (Bio-Rad’s Gel Documentation
System, catalog numbers 170-3742 through 170-3749).
Note: If your are photographing a perpendicular gel, place a ruler on the bottom of the gel
with the 0 mark starting on the low density portion of the gel. This will help determine the
denaturant concentration at which the wild-type and mutant DNA fragments separate.
4. Gels that have been labeled with radioisotopes must be autoradiographed. Carefully place
a 3MM Whatman paper on top of the gel. Gently slide your hand across the paper to
adhere the gel to the paper and to remove any air bubbles. Flip the gel over and place
Saran Wrap™ plastic wrap evenly on top of the gel without creating any bubbles. This
helps to keep the gel intact as well as to prevent any contamination to the gel dryer. Dry
in a gel dryer for about 30–60 minutes at 60 °C.
5. Expose the gel to film and develop film.
29
Section 8
Troubleshooting
Always confirm that the line voltage is correct for the D GENE system.
8.1 Equipment
Problem
Controller
No display on power up
Buffer Circulation
No buffer circulation
Buffer Heating
Can not preheat buffer
Warm-up time too long
Cause
Solution
Burnt fuse
Faulty main switch
Broken wire
Controller malfunction
Replace fuse
Replace main switch
Replace wire
Replace controller
Buffer level too low
Pump failure
Faulty pump switch
Clogged tubing or fitting
Broken inlet tube
Add buffer up to fill level
Replace pump
Replace pump switch
Replace tubing or fitting
Confirm that the inlet tube
extends about 4.5” from cover
Buffer level too low
Level switch malfunction
Cartridge heater burnt
Thermal fuse burnt
Solid state relay damaged
Wrong controller setting
Add buffer to ‘Fill’ level
Replace level switch
Replace cartridge heater
Replace thermal fuse
Replace solid state relay
Set controller parameter
according to controller
instruction
Replace controller
Repair wiring
Add buffer to ‘Fill ‘ level
Replace cartridge heater
Replace solid state relay
Set controller parameter
according to controller
instruction
Place Loading lid on system
Controller malfunction
Loose wiring
Buffer level too low
Cartridge heater burnt
Solid state relay damaged
Wrong controller setting
Loading lid not on system
Non-uniform Temperature
Stir bar not rotating
Damaged motor
Loose set screws at stir bar
Stir bar interferes with
gel sandwiches
Bent stir bar
Buffer level too low
Broken belt
30
Replace motor
Tighten set screws
Maximum thickness of
gel is 1.5 mm
Replace stir bar
Fill to recommended level
Replace belt
Problem
Excessive noise
Excessive noise
during run
Cause
Solution
Worn fan
Damaged pump
Pump touches cell cover
Replace fan
Replace pump
Move pump by shortening
tubing
Replace inlet tube assembly
circulation
Maximum thickness of
gel is 0.5 mm
Align cover
Align stir bar in support tank
Broken inlet tube for buffer
Stir bar interferes with
gel sandwiches
Misaligned core or cover
Stir bar not engaged in
support at tank properly
Casting gels
Glass plate cracked
Perpendicular gradient
Gel solution leaks
Excessive force at
thumb screws
Apply about one turn to thumb
screw after it touches glass
Not sufficient pressure
on comb gasket
Make sure Pressure Clamp
screws are turned one and
one half turns
Bad or wrong comb gasket
Make sure correct comb
gasket is used
Check alignment at bottom of
glass sandwiches, use alignment
slot at casting stand
Use spacers and comb of
same thickness
Perpendicular gradient
Misalignment of spacers
and glass plates
Different thickness of
spacers and comb
Poor, dirty inlet fitting
or missing O-ring
Loose stopcock
No air vent plug
Misaligned comb gasket
Damaged or non
Bio-Rad glass plates
Damaged or dirty spacers
or combs
Poor result
No electrophoresis
Bad cables
Corroded banana plugs
Broken electrodes
31
Replace fitting
Tighten stopcock/injection
connection
Use plug at vent and close it
during gel polymerization
Ensure comb gasket notches
are against spacer notches
Replace with Bio-Rad glass
plates only
Replace spacers or combs
Replace cables
Repair banana plugs
Repair electrodes
8.2 Applications
Problem
Solution
Perpendicular DGGE
Only a single band is seen
in the “S” curve when at
least two bands are expected.
1. Mix normal and mutant
DNA prior to the run.
2. Check PCR reaction products for mutant and normal
DNA by sequencing or restriction digestion.
Hard to visualize heteroduplex and homoduplex
DNA bands.
1. Increase DNA loading.
Unknown faint bands
1. Impurity or contamination of PCR product.
Poor Gradient.
“S” curve not fully seen.
1. Make sure gradient delivery system
is working properly. See instructions.
2. Increase upper gradient concentrations.
Parallel DGGE
Normal and mutant
DNA unresolved.
1. Increase or decrease run time.
2. Poor gradient made. Re-cast gradient gel.
3. Recalculate from perpendicular gel or run a time course gel.
CDGE
Curved band on side lane.
1. Leaking current due to gel spacer problem. Check that
side clamps are fully tightened and correct spacer thickness
are used.
Air bubbles in gel.
1 Clean glass plates.
Fuzzy DNA bands.
1. Gel in well. Clean wells before use. Make sure clamp over
comb in tightened correctly. Check for correct comb
thickness with spacer thickness.
Smear at top of gel.
1. Probably genomic DNA, this is OK.
Bands don’t migrate far
into gel.
1. Increase run time.
2. Decrease acrylamide concentration.
3. Decrease denaturant concentration.
DNA leaks between wells.
1. Acrylamide not polymerized. Add more TEMED and
ammonium persulfate.
2. Degas acrylamide solution before casting gel.
Streaking or DNA
spikes in gel.
1. Impurities in acrylamide. Filter before use.
Check shelf life date of acrylamide solution.
32
Section 9
Maintenance
Maintenance of Equipment
D GENE system with lid
Remove core and clamps from tank. Replace buffer inside
tank with distilled water, turn pump on for 1–2 minutes to
rinse pump. Remove water from tank.
Core, tank, clamps
Rinse thoroughly with distilled water after use.
Glass plates, spacer, combs
Wash with a laboratory detergent (catalog number is 161-0722),
then rinse with distilled water.
Always inspect the D GENE system and replace any damaged components before use.
Repair damaged parts by Bio-Rad trained personnel with Bio-Rad approved components
only.
The controller retains its tuning parameters in non-volatile memory for 10 years without
power. Use unit at least once every 10 years to retain the setting.
33
Section 10
References
For updated references, please request Bio-Rad’s bulletin 1934.
10.1 Applications in Mutation Detection Electrophoresis
Application
Reference numbers
DGGE
Genes
16S rRNA gene ....................................................................52
ABO blood group polymorphism........................................36
Adenine phosphoribosyltransferase gene (APRT)..............186
Adenomatous polyposis coli gene (APC) ...........................85, 95, 119
Alpha-1-antitrypsin gene (AAT) ........................................132, 138
Amyloid precursor protein gene ..........................................21
Androgen receptor gene.......................................................1, 16, 74, 100, 136
Angiotensinogen gene..........................................................80
Apolipoprotein B gene.........................................................167
Apolipoprotein E gene .........................................................42, 44
Beta-globin gene ..................................................................49, 86, 94, 151, 153, 160, 184,
196, 210, 211
Blue-sensitive opsin gene ....................................................124
Cardiac beta-myosin heavy-chain gene (MHC)..................110
CF chromosomes .................................................................53, 56
CF transmembrane conductance regulator gene .................11, 47, 38, 53, 55, 98, 121, 161,
172
cHa-ras 1 proto-oncogene....................................................155
Clonal antigen receptor gene ...............................................169
Collagen gene (COL3A1)....................................................89
Collagen gene (COL4A5)....................................................58, 66, 107
Collagenase gene .................................................................140
Cytochrome P-450 21-hydroxylase genes (CYP21)...........131
Diabetic nephropathy...........................................................80
Dopamine D2 receptor gene ................................................76
F9 gene .................................................................................174
Factor IX gene......................................................................54, 67, 194
Factor VIII gene ...................................................................34, 48, 63, 90, 109, 142, 148,
182, 183, 187, 190
FAU gene .............................................................................29
Gamma globin gene .............................................................84, 113
Growth hormone receptor gene ...........................................41, 51, 189
Gs alpha-gene.......................................................................9, 31, 32, 81, 108, 139, 159, 170
H2kb DNA ...........................................................................154
HEXA gene ..........................................................................37
HOX2B gene........................................................................75
34
Application
Reference numbers
HPRT gene...........................................................................10, 19, 24, 25, 72, 79, 91, 92,
111, 136, 152, 166, 176, 178,
192, 200
Human acid beta-glucosidase gene .....................................188
Human hypoxanthine guanine
phosphoribosyltransferase gene ..........................................112
Human KRAS2 gene ...........................................................60
Human serotonin receptor gene...........................................39
Human thyroid hormone receptor-beta gene (hTR beta) .......120
Hypertension ........................................................................135
Insulin receptor gene............................................................80, 104, 105
K-ras gene ............................................................................6, 13, 64, 73, 82
Low density lipoprotein receptor gene (LDLR)..................26, 99
Mitochondrial DNA.............................................................57, 127
Na+/H+ antiporter gene (APNH) ........................................135, 168, 173
N-ras gene ............................................................................13, 68, 69, 73
NS gene ................................................................................208
Ornithine aminotransferase gene (OAT).............................71, 102, 103
p53 .......................................................................................8, 15, 17, 25, 40, 59, 60, 88, 122,
141, 163, 171
Phenylalanine hydroxylase gene .........................................5, 50
Phosphofructokinase (PFK) gene ........................................65
Pneumocystis carinii gene ...................................................106
Porphobilinogen deaminase gene (PBG) ............................97, 130
Prion protein gene (PrP).......................................................2, 68, 143
Protein C gene......................................................................20, 46, 133
Proteoglycan core protein ....................................................117
PTH gene..............................................................................125
RB1 gene..............................................................................12, 22, 116
Rhodopsin gene....................................................................145, 150
Scalloped gene .....................................................................165
Serotonin receptor gene .......................................................33
Thyrotropin receptor gene ..................................................32
Type II procollagen gene (COL2A1) ..................................4, 23, 43, 62, 197
von Willebrand factor gene..................................................87, 144
Disease
Acute intermittent porphyria (AIP) .....................................97, 130
Albright hereditary osteodystrophy .....................................31, 170
Alport syndrome ..................................................................58, 107
Alport syndrome ..................................................................66
Alzheimer’s disease .............................................................21
Androgen insensitivity syndrome (AIS)..............................74
Autosomal dominant retinitis pigmentosa...........................145, 150
Beta-thalassemias.................................................................49, 86, 151, 160, 184
35
Application
Reference numbers
Breast cancer ........................................................................141
Cancer, general.....................................................................22, 59
Cataracts/retinal detatchment...............................................23
Colorectal adenomas/tumors ...............................................8, 17, 95, 163
Coronary heart disease.........................................................167
Creutzfeldt-Jakob disease ....................................................2, 143
Cystic Fibrosis......................................................................3, 11, 35, 47, 53, 55, 98, 121,
121, 161, 172
Ehlers-Danlos syndrome......................................................89
Familial adenomatous polyposis (FAP) ..............................95, 119
Familial hypercholesterolemia (FH)....................................26, 99
Familial isolated hypoparathyroidism (FIH).......................125
Fetal haemoglobin (HPFH)..................................................113
Gaucher disease (GD)..........................................................188
Generalized resistance to thyroid
hormone syndrome (GRTH) ................................................120
Generalized recessive dystrophic
epidermolysis bullosa (RDEB) ...........................................140
Gout......................................................................................200
Gyrate atrophy (GA) ............................................................71, 102, 103
Haemoglobinopathies ..........................................................14
Hemoglobin cocody variant.................................................27
Hemophilia A.......................................................................45, 48, 63, 90, 109, 128, 142,
148, 182, 183, 187
Hemophilia B Leyden..........................................................54, 194
Hepatocellular carcinoma ....................................................15, 88, 64
HIV-1 ...................................................................................18
Hypertrophic cardiomyopathy.............................................110
Inherited hemoglobinopathies .............................................101
Laron syndrome ...................................................................41, 51, 189
Lung tumour.........................................................................13, 73, 171
Lymphoid neoplasia.............................................................169
Male Pseudohermaphroditism .............................................16
McCune-Albright syndrome (MAS) ...................................108, 139
Medulloblastoma..................................................................122
Mitochondrial disease ..........................................................127
Myoclonic Epilepsy and Ragged-Red Fibres (MEFFR).........57
Nanomelia ............................................................................117
Non-insulin-dependent diabetes mellikus (NIDDM)..........104, 147
Ornithine transcrbamylase deficiency (OTC) .....................179
Osteoarthritis ........................................................................62
Osteochondrodysplasia ........................................................43
Pancreatic tumors.................................................................29, 82
Parathyroid tumors...............................................................29
Phenylketonuria ...................................................................5, 50
36
Application
Reference numbers
Pituitary tumors....................................................................9, 29
Prion disease.........................................................................68
Prostate cancer ....................................................................100
Pseudohypoparathyroidism .................................................81
Retinoblastoma.....................................................................12, 116
Schizophrenia.......................................................................76
Scrapie disease .....................................................................68
Stickler Syndrome................................................................4, 23
Stomach cancer ....................................................................6
Tarui disease.........................................................................65
Tay-Sachs disease ................................................................37
Testicular cancer .................................................................60
Thrombosis...........................................................................46
Thyroid tumor ......................................................................32
Tritanopia .............................................................................124
Type II hereditary protein C deficiency ..............................20
Type IIA von Willebrand disease (vWD) ...........................87
Type IIB von Willebrand disease (vWD)............................144
Wagner syndrome ................................................................23
Other Applications
Addition of nontemplated nucleotides ................................7
Animal population analysis .................................................127
Assessing exposure to environmental carcinogens .............72
Atomic bomb survivors analysis .........................................134
Bantu beta S haplotype ........................................................84
Celtic population analysis....................................................35
Chinese population analysis ................................................184
Conformational transitions of DNA ....................................93, 199, 203
Drosophila recombination analysis .....................................191, 165
Effect of methylation on melting behavior..........................157, 203
Fidelity of DNA polymerase from Pfu................................25
Genetic counseling...............................................................121
Genomic DNA .....................................................................98
Influenza virus RNA molecules ..........................................208
Japanese population analysis ...............................................67, 153
Lipoproteins .........................................................................146, 175, 205
Loss of heterozygosity .........................................................6, 22, 60, 122, 141
Microbial genome size determination .................................193
Microbial population analysis..............................................52
Molecular weight determination of proteins .......................204
Mutational effect of exposure to ENU ................................91
Mutational hot spots.............................................................181
Mutations induced by Thermococcus litoralis ....................19
37
Application
Reference numbers
Natural population analysis .................................................118
Polymorphism detection ......................................................123, 126
Prenatal diagnosis/carrier testing.........................................14, 49, 56, 101, 128, 138, 158,
172, 194
RNA molecular mutation detection.....................................195
Sardinian population analysis ..............................................128
Sexual orientation ................................................................1
Structural analysis of nucleic acids......................................114
UV induced mutation analysis.............................................152
X-ray induced mutations......................................................10, 40
Techniques
Comparison of mutation detection technologies.................22, 77, 137, 158, 188, 190
Fidelity of various DNA polymerases.................................78, 136, 149, 185
GC-clamping........................................................................26, 15, 13, 28, 46, 47, 59, 61, 64,
70, 88, 99, 123, 145, 154, 170,
177, 183, 188, 196, 210, 211
Genomic DGGE...................................................................164
MELT-MAP program..........................................................61, 74, 125, 182, 202
Psoralen-modified oligo primers .........................................28
Restriction fragment melting polymorphisms ....................129
..............................................................................................(RFMP)
Minisatellite allele analysis..................................................156
Southern transfer ..................................................................197
DNA Thermodynamics........................................................232, 233, 234
TGGE ...................................................................................213, 214
CDGE
Genes
HPRT gene...........................................................................224, 230
p53 gene ...............................................................................215, 216, 218, 219, 220, 221,
222, 226, 227, 228
RB1 gene..............................................................................218, 223, 225
Disease
Breast cancer ........................................................................216, 219, 221, 223, 226
Colon cancer ........................................................................223, 225
Gastric cancer.......................................................................227
Lung cancer..........................................................................223
Testis cancer.........................................................................217, 222
Other Applications
Icelandic population analysis...............................................219
Loss of heterozygosity (LOH).............................................218, 221, 222
Techniques
Comparison to other mutation detection methods ..............218, 220, 222, 229
38
10.2 Mutation Detection Electrophoresis References
Denaturing gradient gel electrophoresis (DGGE)
1.
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39
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40
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Chromosome Cancer, 6 (2), 92–97 (1993).
223. Hovig, E., Smith-Sorensen, B., Gebhardt, M. C., Ryberg, D., Lothe, R. and Borresen, A. L., Genes
Chromosome Cancer, 5 (2), 97–103 (1992).
224. Smith-Sorensen, B., Hovig, E., Andersson, B. and Borresen, A. L., Mutat. Res., 269 (1), 41–53
(1992).
46
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), 1100–1108 (1992).
226. Borresen, A. L., Andersen, T. I., Garber, J., Barbier-Piraux, N., Thorlacius, S., Eyfjord, J., Ottestad,
L., Smith-Sorensen, B., Hovig, E. and Malkin, D., et al., Cancer Res., 52 (11), 3234–3236 (1992).
227. Seruca, R., David, L., Holm, R., Nesland, J. M., Fangan, B. M., Castedo, S., Sobrinho-Simoes,
M. and Borresen, A. L., Br. J. Cancer, 65 (5), 708–710 (1992).
228. Borresen, A. L., Hovig, E., Smith-Sorensen, B., Malkin, D., Lystad, S., Andersen, T. I ., Nesland,
J. M., Isselbacher, K. J. and Friend, S. H., Proc. Natl. Acad. Sci. U S A., 88 (19), 8405–8409 (1991).
229. Hovig, E., Smith-Sorensen, B., Brogger, A. and Borrensen, A. L., Mutat. Res., 263 (1), 61 (1991).
230. Hovig, E., Smith-Sorensen, B., Brogger, A. and Borresen, A. L., Mutat. Res., 262 (1), 63-71 (1991).
231. Borrensen, A. L., personal communication.
DNA Thermodynamics
232. Lerman, L. S., Fischer, S. G., Hurley, I., Silverstein, K. and Lumelsky, N., Ann. Rev. Biophys.
Bioeng. 13 399-423 (1984).
233. Poland, D., Biopolymers, 13, 1859-1871 (1974).
234. Gotoh, O. and Tagashira, Y., Biopolymers, 20, 1033-1042 (1981).
47
Section 11
Systems, Accessories, and Reagents for Mutation
Detection Electrophoresis
For updated prices in the U.S., please request Bio-Rad’s bulletin 1935. For specifications,
please request Bio-Rad’s bulletin 1936.
Catalog
Number
Product Description
170-9060
D GENE Complete System, 16 cm, 120 V
170-9061
D GENE Complete System, 16 cm, 100 V
170-9062
D GENE Complete System, 16 cm, 220/240 V
170-9063
D GENE Complete Mini System, 10 cm, 120 V
170-9064
D GENE Complete Mini System, 10 cm, 100 V
170-9065
D GENE Complete Mini System, 10 cm, 220/240 V
170-9006
Converter Kit to 16 cm, D GENE system
170-9007
Converter Kit to 10 cm, D GENE system
170-9008
Comb, 16 well, 0.75 mm, D GENE system
170-9009
Comb, 16 well, 1.00 mm, D GENE system
170-9010
Comb, 16 well, 1.50 mm, D GENE system
170-9011
Glass Inner, 10 cm, 2, D GENE system
170-9012
Glass Outer, 10 cm, 2, D GENE system
170-9013
Glass Inner, 16 cm, 2, D GENE system
170-9014
Glass Outer, 16 cm, 2, D GENE system
170-9016
Tubing Kit, luer inlet fitting, 2, D GENE system
170-9017
Comb, 2 well, 0.75 mm, 10 cm, D GENE system
170-9018
Comb, 2 well, 1.00 mm, 10 cm, D GENE system
170-9019
Comb, 2 well, 1.50 mm, 10 cm, D GENE system
170-9020
Spacer Kit, 16 cm, 0.75 mm, 1 pair, D GENE system
170-9021
Spacer Kit, 16 cm, 1.00 mm, 1 pair, D GENE system
170-9022
Spacer Kit, 16 cm, 1.50 mm, 1 pair, D GENE system
170-9023
Spacer Kit, 10 cm, 0.75 mm, 3 piece set, D GENE system
170-9024
Spacer Kit, 10 cm, 1.00 mm, 3 piece set, D GENE system
170-9025
Spacer Kit, 10 cm, 1.50 mm, 3 piece set, D GENE system
170-9026
Casting Stand Sponge, D GENE system
170-9027
Stand, D GENE system
170-9028
Tank, D GENE system
170-9029
Top Gasket Holder, D GENE system
170-9030
Top Gasket, 1.0 mm, D GENE system
48
Catalog
Number
Product Description
170-9031
Top Gasket, 1.5 mm, D GENE system
170-9032
D GENE Electrophoresis Reagent Kit
170-9033
Replacement Core Gasket, 2
170-9034
MacMelt Software
170-9038
D GENE Control Reagent Kit
170-9039
Casting Stand, D GENE system
170-9040
Clamp Assembly, 10 cm, 1 pair, D GENE system
170-9041
Clamp Assembly, 16 cm, 1 pair, D GENE system
170-9042
D GENE Model 475 Gradient Delivery System
170-9043
Core, D GENE system
170-9044
Comb, 1 well, 0.75 mm, 16 cm, D GENE system
170-9045
Comb, 1 well, 1.00 mm, 16 cm, D GENE system
170-9046
Comb, 1 well, 1.50 mm, 16 cm, D GENE system
170-9047
Filler spacer, for single 7.5 x 10 cm gel, 0.75 mm
170-9048
Filler spacer, for single 7.5 x 10 cm gel, 1.00 mm
170-9049
Filler spacer, for single 7.5 x 10 cm gel, 1.50 mm
170-9053
Replacement belt for stirring bar
170-9054
Tubing kit, Model 475 Gradient Delivery System
170-9055
Pressure Clamp, 10 cm
170-9056
Pressure Clamp, 16 cm
170-- 057
Comb, 32 well, 1.0 mm, 10⁄16 cm, D GENE System
49
Bio-Rad
Laboratories
Life Science
Group
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Telephone (510) 741-1000
Fax: (510) 741-5800
www.bio-rad.com
Bulletin 0000
US/EG
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Phone 089 318 84-177 • Fax 089 318 84-123
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Phone 852-2789-3300 • Fax 852-2789-1257
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Phone 0181 328 2000 • Free Phone 0800-181134 • Fax 01442-259118
00-000
0000
Sig 1200
4000039 Rev B