OPERATING MANUAL Model S2® #21105036

OPERATING MANUAL Model S2® #21105036
OPERATING MANUAL
Model S2® #21105036
Sequencing Gel
Electrophoresis Apparatus
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
S2 SEQUENCING APPARATUS OPERATING MANUAL - TABLE OF CONTENTS
Before You Begin
1.0
Important Information
1.1
Safety Warnings
1.2
Components
1.3
Operating Instructions
Gel Casting
2.0
2.1
Preparation of Glass Plates Using Gel Sealing Tape
2.1.1
Preparation of Glass Plates Using the S2 Casting Clamp
2.1.2
Pouring the Gel Using Gel Sealing Tape
2.1.3
Pouring the Gel Using the S2 Casting Clamp
2.1.4
Electrophoresis
2.2
Pre-electrophoresis
2.2.1
Loading Samples
2.2.2
Electrophoresis
2.2.3
Post-Electrophoresis
2.2.4
Troubleshooting Guide
3.0
References
4.0
Related Products and Replacement Parts
5.0
Care and Handling
6.0
Materials and Care
6.1
General Specifications
6.2
Technical Support and Service
6.3
Instructions for Return Shipment
6.4
Cleaning and Decontamination for Return Shipment
6.4.1
Notice Regarding the Return of Apparatus Products
6.4.2
Warranty
7.0
Warranty
7.1
Declaration of Conformity and CE Mark
7.2
Decontamination Declaration
8.0
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FIGURES
1. S2 Gel Electrophoresis Apparatus Components
2. Taping Glass Plates
3. Assembly Using S2 Casting Clamp
4. Gel Casting Configuration of Sharkstooth Comb
5. Sample Loading Configuration of a Sharkstooth Comb
6. Sharkstooth Comb Sample Loading Technique
7. S2 Repair Components
TABLES
1. Approximate Gel Solution Volume Requirement for Different Gel Thicknesses
2. Recommended Electrophoresis Power Settings for Different Gel Thicknesses
3. Sample Loading Volumes for Model S2 Apparatus Square-Toothed Combs as a Function of
Gel Thickness
MODEL S2® is a registered trademark of Apogee Designs, Ltd.
DELRIN® and MYLAR® are registered trademarks of E.I. DuPont de Nemours & Co.
Tygon® is a registered trademark of Norton Company.
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1.0
BEFORE YOU BEGIN
1.1
IMPORTANT INFORMATION
The Model S2® Sequencing Gel Electrophoresis Apparatus is authorized for laboratory research use
only. It has not been qualified for use in any human or animal diagnostic or therapeutic application.
Use for other than the intended use may be a violation of applicable law.
The Model S2 is designed for vertical polyacrylamide gel separations of the products of dideoxy and
chemical sequencing reactions. The system is useful for preparative purifications of oligonucleotides,
DNA foot printing procedures, ribonuclease protection assays, and sequence determinations of up to
250 to 350 bases. This manual provides operating instructions for the Model S2 Apparatus.
If the product is used in a manner not specified by Apogee, the protection provided by safety features
of the product may be impaired. Please carefully follow the manual’s instructions. Do not alter
equipment or operate with broken components. Failure to adhere to these directions could result in
personal and/or laboratory hazards as well as invalidate the equipment warranty.
1.2
SAFETY WARNINGS

DANGER! HIGH VOLTAGE! This system requires a 1,000 to 3,000 VDC power supply for
operation and is, therefore, a source of high voltage. The power supply should have
earth/ground leakage protection and open-circuit sensing. Although equipped with a safety
interlock system, this equipment must always be operated with extreme caution. Careless
handling can result in possibly fatal electrical shock.

This apparatus should always be operated with caution. Careless handling can result in
electrical shock.

The system should be operated by trained personnel only.

Certain reagents indicated for use in this manual are of a hazardous nature. The researcher is
cautioned to exercise care when handling these reagents. Equipment used in these procedures
(e.g., high voltage power supplies) should be used following the manufacturer’s safety
recommendations.

Always follow the power supply manufacturer’s recommendations for use and follow safety
procedures.

Never operate damaged or leaking equipment. Inspect the apparatus, electrical connections
and power cords prior to use.

For maximum safety, always operate this system in an isolated, low-traffic area, not accessible
to unauthorized personnel.

Before applying DC power to this equipment, make sure that all electrical connections are
secure and that power cords show no signs of damage.

Always turn off the DC power source before disconnecting DC power cords from the sequencing
apparatus. Disconnect power cords from the power source first, and then from the apparatus.
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1.3 COMPONENTS
The Model S2 Apparatus is designed for easy assembly and reliable performance. Please refer to Figure
1 to identify the following principal features and components:

Electrophoresis apparatus with upper buffer chamber, buffer chamber drain valve, silicone
gasket, removable lower buffer tray, integral gel clamps, and safety lids

One pair of 122 cm (48”) red and black power cords

One pair of glass plates (one short, one long)
Long – 333 mm (13.12”) wide x 419 mm (16.50”) long
Short – 333 mm (13.12”) wide x 394 mm (15.50”) long

One pair of 0.40 mm thick vinyl side spacers

One pair of 0.35 mm thick Mylar side spacers

Package of four 0.40 mm thick vinyl sharkstooth combs

Package of two 0.35 mm thick Mylar sharkstooth combs

Package of 12 adhesive-backed foam blocks for side spacers

One roll of high voltage gel sealing tape, 38 mm (1.50”) wide x 66 m (72 yards) long

Instruction manual
Many components are also available separately. Refer to Chapter 5, Related Products for ordering
information. Please read all of the following instructions before using the Model S2 Apparatus. Review
Figure 1 below to identify features and components discussed in these instructions.
Safety lid
Sharkstooth combs
Silicone gasket
Vinyl side spacer
with foam block
Upper buffer drain valve
Long glass plate
Short glass plate
Aluminum plate
Integral gel clamps
Safety lid
Gel supports
DC HV power cords
Removable buffer tray
Figure 1. Model S2 Components
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2.0 OPERATING INSTRUCTIONS
This chapter provides operating instructions for the Model S2 Apparatus. For a list of references
containing formulations of buffers and acrylamide solutions, as well as other applications information
for polyacrylamide gel sequencing and fragment purification, see Chapter 4, References.
2.1 GEL CASTING
Gels can be cast after assembling the glass plates and spacers using Gel Sealing Tape or the S2 Casting
Clamp. These techniques are appropriate for casting polyacrylamide gels.
2.1.1 PREPARATION OF GLASS PLATES USING GEL SEALING TAPE
1. Select a pair of glass plates (one long, one short). Be sure that the edges to be sealed with
tape are free of nicks or chips.
2. Clean the glass plates thoroughly with a nonabrasive detergent and a plastic scouring pad.
The cleaning solution should not leave a soap residue when rinsed thoroughly. Avoid
scratching the surface of the glass plates.
Note: Never use steel wool to scrub the glass plates.
3. Rinse the glass plates thoroughly with deionized water and wipe dry.
4. If desired, treat the inside (acrylamide contact) surface of one or both glass plates (usually
the short plate) with a siliconizing reagent, following the manufacturer’s instructions.
5. Immediately before assembling the glass plate ‘sandwich’, rinse the inside surfaces with
ethanol and wipe them dry with a lint-free paper towel. Avoid touching the inside surfaces
with your fingers.
6. Assemble the glass plate sandwich in the conventional manner: place the long glass plate
inside-up on the bench, align the vinyl side spacers at the sides, and place the short glass
plate inside-down on the side spacers. Make sure that the foam blocks at the tops of the
side spacers are snug against the top of the short glass plate and that the ends of the side
spacers are 4 to 7 mm short of the bottom of the assembled plate sandwich.
Note: Before using vinyl side spacers for the first time, attach a foam block at one end of
each spacer. Remove the protective backing from the adhesive side of a foam block, align
the end of the block with the end of the side spacer, and press the block firmly into place.
Foam blocks may be replaced, as needed, using the extras provided with the Model S2
Apparatus.
7. Seal the sides and bottom of the assembled glass plate sandwich with gel sealing tape, as
shown in Figure 2. Tape the sides first (a, b) and then the bottom (c). Extend the tape
around the bottom corners in both directions to ensure an adequate seal. Apply the tape as
smoothly as possible to avoid forming air channels or bubbles along the edges of the glass
plates. Rub the tape firmly onto glass to eliminate air channels and ensure a liquid-tight
seal.
8. Place two accessory spring clips (available separately) along each side near the middle and
bottom of the glass plate sandwich. This will help maintain uniform gel thickness while
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pouring the gel. It is important for the spring clips to be placed over the side spacers only.
Clamping unsupported glass will distort the thickness of the gel.
Figure 2. Taping Glass Plates
2.1.2 PREPARATION OF GLASS PLATES USING THE S2 CASTING CLAMP
The S2 Casting Clamp can be used in place of the gel sealing tape and spring clips to seal the edges of the
glass plates while casting the gel. Assemble the glass plates as before, and place the assembled
sandwich in the casting clamp following the instructions below.
1. Make sure that the inner surface of the casting clamp is clean of all dried acrylamide and
grease.
2. Hold the casting clamp vertically with your thumbs resting at the top of the molded handles
and your index fingers over the molded feet, flex the clamp outward in a bow-like manner
so that the bottom of the clamp can fit onto the bottom of the gel sandwich.
3. Fit the gel sandwich snugly into the bottom of the casting clamp. Start at one corner and
seal towards the other corner.
4. After the gel sandwich is seated into the bottom of the clamp, fit the sides of the clamp onto
the sides of the gel sandwich. Start at the bottom of one side, using your hands to smooth
the clamp onto the gel sandwich. Repeat this for the other side of the gel sandwich.
5. When properly placed, the sides of the casting clamp will extend to within 4-5-mm of the
top of the short glass plate (See Figure 3). If the sides of the clamp should extend further
than this, reposition the sides of the casting clamp so that they are at the top of the short
glass plate.
Glass plates
Figure 3. Assembly using S2 Casting Clamp
Casting clamp
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2.1.3 POURING THE GEL USING GEL SEALING TAPE
1. Prepare the acrylamide gel solution (see Table 1 for the approximate volumes required for
different gel thicknesses).
Note: Most gel formulations allow approximately 10 min before polymerization.
Polymerization time can be extended by reducing the amount of TEMED added.
Warning: Acrylamide is a known neurotoxin. Consult the Material Safety Data Sheet for
more information.
Table 1. Approximate Gel Solution Volume Requirement for Different Gel Thicknesses.
Gel Thickness (mm)
0.2
0.4
0.8
1.6
Wedge (0.4 to 1.2 mm thick)
Volume Required (ml)
35
65
130
260
140
2. Use a large syringe, squirt bottle, funnel, or beaker to pour the gel solution between the
glass plates. Hold the assembled plate sandwich at a 45° angle on one bottom corner so
that the gel solution flows evenly down along the lower side spacer. Maintain a constant,
even flow to reduce the chance of forming bubbles in the solution.
3. As the gel sandwich fills, gradually lower the glass plates until they rest on the bottom edge,
approximately at a 5° angle from the bench. The gel mold should be slightly overfilled to
ensure complete polymerization at the top.
Note: If a bubble forms while the gel is being poured, raise the glass plates into a vertical
position, and either tip the gel solution away from the bubble or carefully tap the plate
sandwich at the bubble to make it rise to the surface. Once all bubbles have been removed,
return the glass plates to their previous position.
4. Before the acrylamide polymerizes, insert a well-forming comb into the top of the gel. If you
are using a sharkstooth comb, insert the flat edge of the comb between the glass plates to a
depth of 2 to 3 mm below the top of the short glass plate (Figure 4). Keep the flat edge
shallow to simplify subsequent reinsertion of the comb in the sample loading (teeth-down)
configuration and to allow loading with a micropipette. Mark one end of the comb so that
you can keep it in the same left-to-right orientation when loading samples later. If you are
using a DELRIN® square-toothed comb (available separately), insert the teeth 4 to 5 mm
below the edge of the short plate.
5. A top clamp and spring clips are recommended for securing the comb/plate sandwich. Place
two spring clips over the edge of the plates to clamp on the comb (Figure 4). Do not place
the pressure points of the spring clip over the side spacer. Place the top clamp over the top
of the glass plates and clamp the center of the top edges of the plate securely against the
comb. Proper placement of the spring clips and top clamp will force the plates against the
comb, ensuring a tight fit of the comb following polymerization. If you use spring clips along
the lower edge of the gel for sealing purposes, clamp them on the side spacers.
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Note: If spring clips are not put in place during polymerization, the comb will not fit tightly,
which increases the possibility of leaks between wells.
Figure 4. Gel Casting Configuration of Sharkstooth Comb
6. Leave the glass plates in their near-horizontal position until the acrylamide has polymerized.
7. Remove the spring clips (and top clamp, if used) and tape. Carefully slide the comb from
between the glass plates.
8. Rinse the top of the gel with electrophoresis buffer to remove any unpolymerized
acrylamide. Rinse the comb with deionized water.
9. If you are using a sharkstooth comb, reinsert the comb between the glass plates with the
teeth toward the gel. Insert the comb until the teeth just make contact with the surface of
the gel (figure 5). Do not allow the teeth to pierce the gel. A very slight indentation of the
gel should be visible when the comb is properly inserted. Do not slide the comb laterally
after it has come in contact with the gel.
2.1.4 POURING THE GEL USING THE S2 CASTING CLAMP
The S2 Casting Clamp allows for the gel to be filled from either the top or the bottom of the assembled
plate sandwich.
To cast the gel from the top, follow the
instructions below making sure that the
bottom fill port is sealed.
1. To fill the gel from the top, use a
large syringe, squirt bottle, funnel,
or beaker to pour the gel solution
between the plate sandwich.
Hold the assembled plate
sandwich at a 25°-35° angle on
Figure 5. Sample Loading
one bottom corner so that the gel
Configuration of Sharkstooth Comb
solution flows evenly down along
the lower side spacer. Maintain a constant, even flow to reduce the chance of forming
bubbles in the solution.
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2. As the gel sandwich fills, gradually lower the casting clamp until it rests on the molded feet,
approximately a 5° angle. The gel mold should be slightly overfilled to ensure complete
polymerization at the top.
3. Before the acrylamide polymerizes, insert a well-forming comb into the top of the gel. If you
are using a sharkstooth comb, insert the flat edge of the comb between the glass plates to a
depth of 2 to 3 mm below the top of the short glass plate (Figure 4). Keep the flat edge
shallow to simplify subsequent reinsertion of the comb in the sample loading (teeth-down)
configuration and to allow loading with a micropipette. Mark one end of the comb so that
you can keep it in the same left-to-right orientation when loading samples later. If you are
using a DELRIN® square-toothed comb (available separately), insert the teeth 4 to 5 mm
below the edge of the short plate.
4. For improved comb sealing, clamp the glass plate sandwich over the comb with a top clamp
over the center of the comb. It is important for the top clamp to be placed over the comb
only. Clamping unsupported glass will distort the thickness of the gel. The casting clamp
does not require the use of accessory spring clips along the sides of the gel sandwich.
5. Leave the glass plates in their near-horizontal position until the acrylamide has polymerized.
6. Remove the top clamp (if used) and carefully slide the comb from between the glass plates.
7. Remove the glass plates from the casting clamp. Pull the casting clamp off the glass plates
starting at the top and working your way down. Take care in that there may be some
unpolymerized acrylamide in the bottom of the casting clamp.
8. Rinse the top of the gel with electrophoresis buffer to remove any unpolymerized
acrylamide. Rinse the comb with deionized water.
9. If you are using a sharkstooth comb, reinsert the comb between the glass plates with the
teeth toward the gel. Insert the comb until the teeth just make contact with the surface of
the gel (Figure 5). Do not allow the teeth to pierce the gel. A very slight indentation of the
gel should be visible when the comb is properly inserted. Do not slide the comb laterally
after it has come in contact with the gel.
To fill the gel from the bottom, assemble the gel plates so that when the casting clamp is laying down on
the molded feet, the short glass plate is on top.
1. Place the assembled gel sandwich on a level surface resting on the molded feet of the
casting clamp.
2. Open the bottom fill port and insert a Luer fitting.
3. Attach a syringe of suitable reservoir to the fitting and slowly fill the gel. Do not interrupt
the filling as this may introduce bubbles into the gel solution.
4. Once the gel is filled, insert the comb as before and allow the gel to polymerize.
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2.2 ELECTROPHORESIS
2.2.1 PRE-ELECTROPHORESIS
1. Before beginning electrophoresis, verify that the sequencing apparatus is in a secure and
level position.
2. Place the gel sandwich in the sequencing apparatus with the short glass plate inward, so
that the foam blocks on the side spacers form a seal with the blue silicone gasket. Rest the
bottom edge of the sandwich on the ribbed gel support blocks in the lower buffer tray.
3. Secure the gel sandwich with the integral gel clamps along the sides of the sequencing
apparatus. Tighten the screw knobs firmly, but do not over tighten.
4. Verify that the upper buffer chamber drain valve is closed, and fill the upper buffer chamber
with approximately 450 ml of electrophoresis buffer. Make sure no buffer is leaking from
the upper buffer chamber. Fill the front chamber of the lower buffer tray with
approximately 450 ml of electrophoresis buffer. Be sure no bubbles obstruct buffer contact
with the lower edge of the gel.
Note: Do not overfill the buffer chambers or allow buffer to make contact with the electrode
mounting nuts.
Warning: No buffer should leak through or around the silicone gasket or down the side of
the gel assembly. Leakage may allow the upper buffer chamber to drain dry or cause
sparking and damage to the apparatus. See Chapter 3, Troubleshooting, for further
information.
5. Close the upper and lower safety lids and connect the DC power cords to the sequencing
apparatus and the DC power supply.
Warning: All banana plug connections must be fully seated to prevent potential sparking
and fire hazards.
6. Before loading samples, pre-electrophorese a gel for 15 to 45 min (see Table 2 for
recommended DC power settings for various gel thicknesses). Best results are achieved with
a gel surface temperature of ~50°C.
Warning: Excessive power will cause the gel to overheat and crack the glass plates.
Table 2. Recommended Electrophoresis Power Settings for Different Gel Thicknesses
Gel Thickness (mm)
0.2
0.4
0.8
1.6
Wedge
Watts (W=V x A)
55
60
65
70
85 to 90
Volts (V)
2,200 to 2,600
1,500 to 1,900
1,100 to 1,200
600 to 700
1,200
Current (mA)
20 to 35
30 to 45
60 to 90
95 to 125
~70
Note: Power conditions were determined for a 50°C surface temperature with 1x TBE buffer.
~0.4 to 1.2 mm thick.
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2.2.2 LOADING SAMPLES
1. Prepare the samples by heating in an appropriate loading buffer to a temperature of 90°C to
100°C for 3 to 5 min and then chilling on ice.
2. At the end of the pre-electrophoresis period, turn off the power supply and disconnect both
DC power cords, first from the power supply and then from the
sequencing apparatus, before opening the upper safety lid.
3. Immediately prior to loading the samples, rinse the wells of the gel
with electrophoresis buffer. Use a Pasteur pipette to wash away urea
that has diffused into the wells.
Note: Not rinsing the wells will prevent samples from layering
properly on the gel surface.
4. Using a sharkstooth comb to form shallow wells (see Section 2.1.3)
allows you to load samples with a micropipette or a drawn-out
capillary pipette (Figure 6). When using the sharkstooth comb
Figure 6. Sharkstooth Comb
provided with the Model S2 Apparatus, load 2 to 3 µl of
Sample Loading Technique
sample per well. When using accessory double-fine
sharkstooth combs, load 1.5 to 2 µl of sample.
5. When using optional square-toothed combs, load samples onto the bottom of the wells with
capillary pipettes. To determine the sample loading volumes of Model S2 Apparatus squaretoothed combs relative to gel thickness, see Table 3.
Table 3. Sample Loading Volumes f or Model S2 Apparatus
Square-Toothed Combs as a Function of Gel Thickness
Number of Teeth
16
Tooth Width (mm)
14.6
20
11.1
32
5.7
Gel Thickness (mm)
0.4
0.8
1.6
0.4
0.8
1.6
0.4
0.8
1.6
Capacity/Well (ul)
25
50
100
19
38
76
10
20
40
Note: All loading volumes are calculated for a comb insertion depth of 5 mm.
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2.2.3 ELECTROPHORESIS
After loading the samples, close the upper and lower safety lids and connect the DC power cords to the
sequencing apparatus and the DC power supply.
Warning: All banana plug connections must be fully seated to prevent potential sparking
and fire hazards.
1. Turn on the power supply and set the voltage or wattage to the proper setting for the gel
(see Table 2 for recommended DC power settings for various gel thicknesses).
2. Monitor the progress of electrophoresis by following the migration of a marker dye front or
by any other preferred method.
3. When electrophoresis is complete, turn off the power supply and disconnect both DC power
cords, first from the power supply, and then from the sequencing apparatus.
2.2.4 POST-ELECTROPHORESIS
1. Open the buffer chamber drain valve to allow the buffer in the upper chamber to drain into
the covered rear chamber of the lower buffer tray. Open the upper safety lid and rinse the
upper buffer chamber with 25 to 50 ml of deionized water to prevent buffer crystallization
in the internal drain tubing.
2. Release the integral gel clamps by loosening the screw knobs and remove the gel sandwich
from the sequencing apparatus. Lay the gel sandwich flat, with the short glass plate on top,
on a piece of absorbent paper.
3. Open the lower safety lid and remove the lower buffer tray from the sequencing apparatus.
The buffer in the open front chamber of the tray will contain any radioactive nucleotides
electrophoresed out of the gel. Dispose of this liquid by pouring away from the drain hole in
the covered rear chamber of the tray. The buffer in the rear chamber (drained from the
upper chamber) may then be discarded separately. After disposing of all buffer, rinse both
chambers of the tray thoroughly with deionized water and place the tray back in the
sequencing unit.
4. Disassemble the gel sandwich by prying off the short (or siliconized) glass plate, working
gently from a bottom corner with a thin spatula.
5. For autoradiography, transfer the gel to a solid backing, such as paper or used film.
6. Rinse combs, side spacers, and glass plates to remove buffer and gel fragments, and dry all
components before storing.
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3.0 TROUBLESHOOTING GUIDE
Some suggestions for resolving common problems are given below. Should these suggestions not
resolve the problem, please call Technical Support (see Section 6.3 for numbers). If the unit must be
returned for repair, also contact our service department, the technical support or your local distributor
for shipping instructions. Please include a full description of the problem.
PROBLEM
Gel Solution leaks from the bottom
SUGGESTED SOLUTION
Lower the angle of the glass plates when pouring the
gel during casting.
Check that the inner surface of the casting clamp is
clean and free of debris and grease. Make sure that
the glass plates are firmly seated against the inner
surface of the casting clamp.
The glass plates crack
The gel is too hot. Use a lower wattage or voltage
setting.
The upper buffer chamber will not drain
Check that the valve knob has been released
sufficiently.
Force water or buffer through the drain outlet with a
syringe or pipette to remove possible dried buffer
residue, airlock, or collapse in the drain tubing.
‘Wiggly’ or ‘slanting’ bands (bands are not
straight lines or parallel to the top edges of
the gel).
Verify that the wells are free of particles and bubbles
before and after loading samples.
Verify that the agarose is completely dissolved before
casting gels.
Remove any particulate matter from the agarose
before casting gels.
Be sure that bubbles are not trapped against the comb
during gel casting.
All bands appear as ‘doublets’ (each band is
represented twice within the same lane).
Concentrate the sample and use a thin (2 to 3 mm)
gel with a thin (1 mm) comb.
Prevent gel movement during photography.
The gel dye front is not straight
Verify that the system is level.
Make sure the system is not in a draft or otherwise
being cooled unevenly.
Verify that the buffer depth in both buffer trays is
sufficient to provide complete, even contact across
the full width of the gel.
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Verify that both electrode carriers are properly
clipped in place.
Make sure that the spring clips are placed over the
side spacers. Distortion of the gel thickness will cause
the dye front to run at different rates across the gel.
Buffer leaks from the upper buffer chamber
Verify that the top integral gel clamps are firmly, but
not overly, tightened. The ribs of the silicone gasket
should be slightly and evenly compressed, but not
wrinkled. First, loosen the integral gel clamps a partial
turn. If the leaking does not stop, try tightening the
clamps carefully.
Verify that the black foam blocks are securely seated
against the short glass plate before casting the gel.
Check the silicone gasket for tears or other damage.
Verify that a pinhole leak or crack has not developed
in the glue joints of the upper buffer reservoir.
Sparks or burn marks appear at edges of
aluminum plate.
Buffer is leaking from the upper buffer chamber. See
preceding comments.
CAUSES OF COMMON PROBLEMS IN SEQUENCING GEL ELECTROPHORESIS
PROBLEM
POSSIBLE CAUSE
SUGGESTED SOLUTION
Blurry bands
Urea diffusing into the wells
After pre-electrophoresis, wash the urea from
the wells before loading the samples.
Formamide in sample buffer degraded
Remake sample buffer with fresh formamide.
Excessive salt in the sample
Precipitate DNA with ethanol and ammonium
acetate.
Radiolabel properties lead to scattering
of signal
Try 35S- or 33P-labeled dNTP’s instead of 32P
labeled dNTP'.
Label the sequencing primer with T4
polynucleotide kinase instead of
internal labeling.
Use a shorter exposure.
Use cassette that assures tight contact of gel to film.
Do not use an intensifying screen.
Gel was overloaded
Load less sample.
Urea diffused out of gel
Do not store precast gels under electrophoresis
buffer overnight in the sequencing unit.
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PROBLEM
POSSIBLE CAUSE
SUGGESTED SOLUTION
Smeared
bands
Sample electrophoresing with the ion
in front
Pre-electrophorese gel until the current starts
to drop (generally > 1/2 hour).
Gel temperature too high
Do not electrophorese gel on constant
current. Use constant power to
maintain gel at 45–50°C. (Use a surface
thermometer during electrophoresis.)
Sample electrophoresing at the
surface of the acrylamide, instead
of through gel
Thoroughly rinse residual siliconizing agent
off glass plates. Ethanol wipe before using.
Thoroughly wash new glass plates with a strong
lab soap before using.
Wavy bands
Formamide in sample buffer degraded
Remake sample buffer with fresh formamide.
Excessive salt in the sample
Precipitate DNA with ethanol and ammonium
acetate.
Surface of the gel did not polymerize
evenly.
Recast gel with extra acrylamide at the top of
the gel.
Clean the flat edge of the sharkstooth comb.
Glass plates
crack
Sharkstooth combs punctured
the surface of the gel
Insert sharkstooth comb only to the point of
dimpling the surface of the gel.
Gel is too hot
Electrophorese with constant voltage or
power, not constant current.
Do not exceed settings that yield a gel surface
temperature of 45–50°C.
Lack of
Limitations in the resolving capabilities
resolution in of acrylamide.
the upper half
of the gel
Capsules used to make 10% ammonium
persulfate solution.
Use a buffer gradient gel.
Use wedge spacers.
Do a double loading.
Use ammonium persulfate powder to make
solution.
Alternatively, remove the ammonium
persulfate from the capsule before dissolving
(12).
Smiling gels
Gel thickness is not uniform
(Corresponding
bands in outer lanes
electrophorese
slower than
inner lanes)
Allow the gel to polymerize at a 5° angle
instead of flat.
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
PROBLEM
POSSIBLE CAUSE
SUGGESTED SOLUTION
Gel temperature is not uniform
Use an electrophoresis unit with an aluminum
plate to disperse heat evenly.
Frowning gels Improper clamping during casting
(Corresponding
bands in outer
lanes electrophorese
faster than
inner lanes).
Apply the pressure points of the binder clips
only over the spacer.
Polymerize the gel at an angle of <10°.
Do not over tighten clamps on the apparatus.
Improper clamping of the gel into the
electrophoresis chamber.
Lanes curve
Improper gel casting
outward toward
bottom of the gel.
Make sure that spacers are pushed against
the polymerized acrylamide.
Do not use bottom clamps without a bottom
spacer.
Do not pull plates together while taping the gel.
Bands are not Gel sandwich is not level
even from
lane to lane.
Make sure glass plates are seated
flat in the electrophoresis unit.
Make sure the gel electrophoresis unit is
level.
4.0 REFERENCES
1. Anderson, S. (1981) Nucl. Acids Res. 9, 3015.
2. Bankier, A.T., Weston, K.M., and Barrell, B.G. (1987) Methods Enzymol. 155, 51.
3. Life Technologies, Inc. (1985) M13 Cloning/Dideoxy Sequencing Instruction Manual.
4. Blakesley, R. (1983) Focus® 5:1, 1.
5. Johnson-Dow, L., Mardis, E., Heiner, C., and Roe, B.A. (1987) BioTechniques 5, 754.
6. Kuebbing, D. (1983) Focus 5:2, 1.
7. Martin, R. (1987) Focus 9:1, 8.
8. Maxam, A.M. and Gilbert, W. (1980) Methods Enzymol. 65, 499.
9. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Nat. Acad. Sci. USA 74, 5643.
10. Smith, A.J.H. (1980) Methods Enzymol. 65, 560.
11. Tullius, T.C., Dombroski, B.A., Churchill, M.E.A., and Kam, L. (1987) Methods Enzymol. 155,
537.
12. Hartley, J. and Xu, L. (1994) Focus 16, 52.
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
5.0 RELATED PRODUCTS AND REPLACEMENT PARTS
Accessory
Description
Catalog #
Spacer Sets
Includes two side spacers and 12 foam blocks
0.19 mm thick (Mylar)
0.35 mm thick (Mylar)
0.4 mm thick
0.8 mm thick
1.6 mm thick
21105309
21105366
21108014
31109010
31109028
Individual Bottom Spacers
0.4 mm thick
0.8 mm thick
1.2 mm thick
1.6 mm thick
21105077
21105069
21105325
21105051
Wedge Spacer Set
Complete with two 0.4 to 1.2 mm thick
side spacers and 12 foam blocks
21107016
Silicone Spacer Foam Blocks
Package of 12
21965017
16 tooth Machined Analytical DELRIN Comb
0.4 mm thick
0.8 mm thick
1.6 mm thick
21035043
21035050
21035068
20 tooth Machined Analytical DELRIN Comb
0.4 mm thick
0.8 mm thick
1.6 mm thick
21035076
21035084
21035092
32 tooth Machined Analytical DELRIN Comb
0.4 mm thick
0.8 mm thick
1.6 mm thick
21035100
21035118
21035126
14 cm Mylar Sharkstooth Comb
25-pt., 0.19 mm thick (pkg. of 6)
25-pt., 0.35 mm thick (pkg. of 6)
21105317
21105341
14 cm Vinyl Sharkstooth Comb
25-pt., 0.4 mm thick (pkg. of 6)
21045018
14 cm Vinyl Double fine Sharkstooth Combs
49-pt., 0.4 mm thick (pkg. of 4)
21045026
28 cm Mylar Sharkstooth Comb
62-pt., 0.35 mm thick (pkg. of 2)
21035134
28 cm Vinyl Sharkstooth Combs
50-pt., 0.4 mm thick (pkg. of 2)
21046016
Glass Plates (pair of short and long)
Precision, flat, clean edges
Long – 333 mm (13.12”) wide x 419 mm (16.50”) long
Short – 333 mm (13.12”) wide x 394 mm (15.50”) long
11034014
S2 Casting Clamp
21105432
Package of 1
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
HV Gel Sealing Tape (1.5 in.  72 yd.)
1 roll
10 rolls
11032018
11032026
Replacement Part
Description
Catalog #
Power Cord Replacements 1 black & 1 red, 122 cm long
Package of 2
11099025
Silicone Gasket, grey
Kit
21105358
S2 Upper Buffer Pt/Ti Electrode Replacement
Includes all necessary components
Kit
21113014
S2 Lower Buffer Pt/Ti Electrode Replacement
Includes all necessary components
Kit
11114014
Upper Buffer Chamber Banana Plug Replacement
(Includes cap nut, O-ring and Banana Plug)
Kit (see Figure 7)
21105127
Lower Buffer Chamber Banana Plug Replacement
(Includes cap nut, O-ring and Banana Plug)
Kit (see Figure 7)
21105101
Gel Clamp Replacement
Kit
11958352
Pt/Ti electrode wire
Flat washer
Wire carrier
Acorn nut
Banana plug
Figure 7. S2 Repair Components
Gel clamp
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
6.0 CARE AND HANDLING
6.1 MATERIALS AND CARE
The S2 is the world standard manual sequencing apparatus. Every S2 apparatus is machined and
fabricated from high quality aluminum, ABS, acrylic and polycarbonate plastic. Acrylic and ABS both
have very good heat, impact, and chemical resistance but will not withstand autoclaving.
Caution: Both electrodes are made from Pt/Ti ire for durability. Use care when cleaning this
apparatus to prevent breakage of the electrodes because they are not warranted against breakage.
All components may be washed with water and a detergent. To remove grease and oils, use a hexane,
kerosene, or aliphatic naphtha. Never use abrasive cleaners, window sprays, or any fluid that may
contain toluene, methylene chloride, phenol, acetone, benzene, halogenated hydrocarbon solvents, or
undiluted laboratory alcohols.
The following simple, routine inspection and maintenance procedures will help ensure both the safety
and the performance of the Model S2 Apparatus. For ordering information for replacement parts, refer
to Chapter 5. For replacement parts, call your distributor or Apogee Technical Support.
1. Because of the high voltages that may be used, inspect electrical connections and power cords
often. If power cords show any signs of wear or damage (e.g., cracks, nicks, abrasions, melted
insulation or bare wire), replace immediately.
2. If the banana plug connectors to the unit or the power supply do not exhibit reasonable friction
or can be rocked easily, replace the plugs or power cords.
3. Examine the electrode banana plugs and connection nuts to ensure that they are free of
corrosion or they may offer higher resistance thus heating up and risking sparks and fire. If
connection nuts are corroded, they should be replaced.
4. Regularly inspect the silicone gasket for cuts or tears and replace, as needed.
5. After each use, rinse the aluminum plates thoroughly with deionized water to remove salts and
urea. Failure to keep plates properly clean may affect performance and introduce a potential
electrical hazard.
6.2 GENERAL SPECIFICATIONS
Type
Dimensions (W × L x H)
Weight
Gel Dimensions
Maximum gel thickness
Voltage & Current Range
Electrode material
Operating Temperature Range
Construction
Model S2
42.2  21.6  44.5 cm
6.32 kg
31 x 38.5 cm
1.6 mm
2,000 VDC Max at 0-125 mA, 0.5 A Max
Pt/Ti wire
4-30°C (non-condensing atmosphere)
Flame retardant ABS, acrylic, aluminum, silicone
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
6.3 TECHNICAL SUPPORT AND SERVICE
Should you have any problems with this unit, please contact:
Apogee Designs, Ltd.
Attn: Electrophoresis Support
101 Kane Street
Baltimore, MD 21224 USA
Phone:
Fax:
Email:
443.744.0368
9 to 5PM EST, Monday through Friday
410.633.3666
[email protected]
6.4 INSTRUCTIONS FOR RETURN SHIPMENT
IMPORTANT: Before sending the unit back to us, it is absolutely necessary to call our Technical
Support department to get authorization to return products!

Return only defective devices. For technical problems which are not definitively recognizable as
device faults please contact Apogee Technical Support.

Use the original box or a similarly sturdy one.

Label the outside of the box with CAUTION! SENSITIVE INSTRUMENT!

Please enclose a detailed description of the fault and when, or how, the problem occurred.
Important: Clean all parts of the instrument from residues and of biologically dangerous, chemical and
radioactive contaminants. Please include a written confirmation (use the respective Decontamination
Declaration/Certificate following in Section 8 that the device is free of biologically dangerous and
radioactive contaminants in each shipment. If the device is contaminated, it is possible that Apogee will
be forced to refuse to accept the device. The sender of the repair order will be held liable for possible
damages resulting from insufficient decontamination of the device.
Please enclose a note which contains the following:
1. Sender's name and address and,
2. Name of a contact person for further inquiries with telephone number.
6.4.1 CLEANING AND DECONTAMINATION FOR RETURN OF PRODUCTS
Use the original product packaging whenever possible, to avoid damage to the unit being returned. All
returned material must be cleaned and decontaminated prior to shipping. The components of
apparatus products are fabricated from a variety of materials including: ABS, acrylic, vinyl, glass, silicone,
aluminum and stainless steel.
Please clean any unit or product to be returned using the following three step procedure.
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
STEP 1: GENERAL CLEANING PROCEDURE
For materials not contaminated with biological or radiological substances, components may be gently
washed with water and a non-abrasive detergent, and rinsed with deionized water. Dry using a soft
cloth, paper towel or allow to air dry. A light application of hexane, kerosene, or aliphatic naphtha will
remove grease.
To prevent surface damage, never use abrasive cleaners, window sprays or scouring pads to clean these
products. Avoid excessive exposure to UV light, phenol, acetone, benzene, halogenated hydrocarbon
solvents, or undiluted alcohols because they may cause crazing.
STEP 2: BIOLOGICAL CLEANING PROCEDURE
Using a solution of either 5% household bleach in water or 70% ethanol in water, wipe down the
apparatus using a clean cloth or sponge. Neutralize the solution by wiping the surface with a mild,
nonabrasive detergent and rinse well with water.
STEP 3: RADIOLOGICAL DECONTAMINATION PROCEDURE
To meet various regulatory and safety standards, please follow the decontamination procedure given
here if radioactive materials are used with this product or are used in the vicinity of where this
apparatus has been used or stored.
WARNING: We cannot and will not accept return of products that are contaminated with any
radioactivity.
For beta emitting isotopes such as 32P, use a GM-type radioactivity meter calibrated in counts per
minute (CPM) to determine the background readings for your work area. Wearing latex gloves, survey
the unit to be returned with the GM meter. If any part of the unit is found to show readings higher than
background, wash the area using Radiacwash© (Atomic Products Corp.) and paper towels, or another
similar commercially available detergent. If none are available, a mild detergent or a Formula 409© type
solution will do. As you clean, discard liquid and solid waste (gloves and paper towels) according to your
local and institutional regulations for radioactive material disposal. Continue washing until the GMmeter reading for the contaminated area(s) is equal to or below background.
To decontaminate units where a GM-meter is not as useful for detection, as with 'H, or "S, it will be
necessary to perform swipes of the unit and detect using a scintillation counter. The unit should be dry.
Wipe surfaces with dry paper circles (these are commercially available or you can make your own).
Areas can be charted, so that individual swipes can be done on different surfaces to better isolate areas
of contamination.
Swipes should be placed into individual scintillation vials with an appropriate floor and then analyzed on
a properly programmed scintillation counter. If contamination above 100 disintegrations per minute
dpm/100cm2 (dpm=CPM/efficiency) is found, wash the area as described above in 32P decontamination.
After cleaning the area, swipe it a second time to determine the amount of contamination remaining. If
the area still has greater than 100 dpm/cm2, continue the cycle of swipes and washing until you achieve
a reading of less than 100 dpm/cm2.
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
Once the unit has been determined to be radiation free (<100dpm/cm2) remove all the hazardous and
radioactive labels from the unit. If the labels cannot be removed, deface them. Failure to do so may
result in a significant delay or refusal of repair. If your unit has non removable contamination
(detectable with a GM-meter and not with paper swipes, or detectable with paper swipes but after
continued washing the dpm/cm2 remains constant and above 100) of a short half life isotope such as 32P,
it may be stored for ten half lives of isotopic decay and the decontamination procedure repeated.
Note: Units contaminated with non removable, long half life isotopes may not be returned.
If questions still persist, please contact:
Apogee Designs, Ltd.
Attn: Electrophoresis Support
101 Kane Street
Baltimore, MD 21224 USA
Phone:
Fax:
Email:
443.744.0368
9 to 5PM EST, Monday through Friday
410.633.3666
[email protected]
6.4.2 NOTICE REGARDING THE RETURN OF APPARATUS PRODUCTS
US Federal Regulations
In order to comply with US federal regulations and to protect the health and safety of employees, it is
imperative that all customers read this notice and adhere to the requirements regarding the return of
apparatus products. The US Department of Transportation, the Department of Health and Human
Services, and the Nuclear Regulatory Commission have strict regulations on the shipment of hazardous
materials (49 CFR Part 173) including etiologic agents (49 CFR Part 173 and 42 CFR Part 72) and
radioactive materials (CFR 49 Part 173 and 10 CFR Part 20).
German Law
To comply with German law (i.e. §71 StrlSchV, §17 GefStoffV and §19 ChemG) and to avoid exposure to
hazardous materials during handling or repair, completion of this form is required before equipment
leaves your laboratory. When equipment is returned for repair, evaluation, credit or exchange, the
customer becomes the shipper and must ensure that the item is free of contamination whether
chemical, biological or radioactive. Procedures for decontamination are described above.
Materials received that have not been properly decontaminated or units which do not have hazard
labels (such as ‘caution radioactive materials’) may be decontaminated at the customer's expense
(approximately $350) and may result in delay or refusal of repair. In addition, in the case of radioactive
contamination, Apogee may be required to notify a licensing authority that in turn may be required to
notify the customer's licensing authority.
Please carefully follow the instructions on decontamination and fill out the Decontamination Declaration
that follows. Place the Decontamination Declaration inside the top flap of the box where it can be
immediately noticed by the receiver. Any change to this procedure may result in service delay.
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
7.0 WARRANTY
7.1 WARRANTY
Apogee warrants apparatus of its manufacture against defects in materials and workmanship, under
normal service, for one year from the date of receipt by the purchaser. This warranty excludes damages
resulting from shipping, misuse, carelessness, or neglect and does not include breakage of the
electrodes or crazing from cleaning with solvents that attack ABS or acrylic. Apogee’s liability under the
warranty is limited to the repair of such defects or the replacement of the product, at its option, and is
subject to receipt of reasonable proof by the customer that the defect is embraced within the terms of
the warranty. All claims made under this warranty must be presented to within three years following
the date of delivery of the product to the customer.
This warranty is in lieu of any other warranties or guarantees, expressed or implied, arising by law or
otherwise. Apogee makes no other warranty, expressed or implied, including warranties of
merchantability or fitness for a particular purpose. Under no circumstances shall Apogee be liable for
damages either consequential, compensatory, incidental or special, sounding in negligence, strict
liability, breach of warranty or any other theory, arising out of the use of the product listed herein.
In the interest of bettering performance, Apogee reserves the right to make improvements to the
design, construction, and appearance without notice.
7.2 DECLARATION OF CONFORMITY AND CE MARK
Note: The information outlined in this section applies only to customers located in the European Union
(EU).
This laboratory apparatus is identified with the CE mark. This mark indicates that the product complies
with the following EU Directives and Standards:
APPLICATION OF COUNCIL DIRECTIVE(S):
89/336/EEC
73/23/EEC
Electromagnetic Compatibility
Low Voltage Directive
STANDARDS:
EN 50081-1:1992
EN 50082-1:1992
EN 61010-1:1993
Emissions
Immunity
Product Safety
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
8.0 DECONTAMINATION DECLARATION
RGA Number (IMPORTANT): ___________________________________________________________
Customer Name: ____________________________________________________________________
Institute: ___________________________________________________________________________
Address: ___________________________________________________________________________
TEL #: ____________________________________ FAX #: __________________________________
E-mail: ____________________________________________________________________________
Unit type: _________________________________ Serial number: ___________________________
DESCRIPTION OF PROCEDURES USED TO DECONTAMINATE UNIT (LOOK AT6.4.1)
□ 1. S2 was gently washed with water and a non-abrasive detergent, and rinsed with deionized water.
□ 2. Using a solution of 5% household bleach in water or 70% ethanol in water, the unit was wiped
down using a clean cloth or sponge and neutralized with deionized water.
□ 3. To meet various regulatory and safety standards, the decontamination procedures given in 6.4.1
if radioactive materials were used to decontaminate this product.
This piece of equipment has not been decontaminated. Reason:
□ To the best of my knowledge, unit is free of chemical, biological, or radioactive contamination.
I understand that if the equipment is found to be contaminated, regardless of the signature on this
document, the equipment may be decontaminated at my expense. Also, if the equipment is found to be
contaminated, the response time for repairs will be delayed.
Signature: __________________________________________________________________________
Title: ________________________________________
Date: ________________________________________
Please place completed and signed form inside the box with the equipment where it can
immediately be noticed by the receiver. We appreciate you taking the time to perform
the necessary precautions to ensure that equipment being returned can be safely
handled by our employees.
Apogee Electrophoresis | 101 Kane Street | Baltimore, MD 21224 USA | apogeephoresis.com
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