Apogee Horizon H58 User manual

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INSTRUCTION MANUAL
Horizon H58™#21065040
Gel Casting System

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GEL CASTING APPARATUS INSTRUCTION MANUAL - TABLE OF CONTENTS
Before You Begin 1.0
Important Information 1.1
Components 1.2
Gel casting procedure 2.0
Troubleshooting Guide 3.0
Applications 4.0
Considerations for Agarose Gel Electrophoresis 4.1
Selecting Gel Concentration 4.1.1
Preparing Agarose for Gels 4.1.2
Preparing Samples and Loading the Gel 4.1.3
Ethidium Bromide Staining of Double-Stranded DNA 4.2
Gel Photography 4.3
Related Products 5.0
H48 Accessories 5.1
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
Decontamination Declaration 8.0
Horizon ®is a registered trademark of Apogee Designs, Ltd.
Delrin® is a registered trademark of E.I. duPont de Nemours & Co.

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1.0 BEFORE YOU BEGIN
1.1 IMPORTANT INFORMATION
The Horizon H58™Gel Casting System is a stand-alone device for casting gels for the H58 horizontal gel
electrophoresis unit. This system can be used to cast gels while the electrophoresis unit is being used or
to cast multiple gels in advance.
For operating procedures for the Horizon H58 Apparatus, as well as general guidelines, practical
applications, and suggestions for agarose gel electrophoresis, please refer to the apparatus instruction
manuals. These systems have 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.
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.
1.2COMPONENTS
The H58 Gel Casting System is engineered for durable performance and easy storage. The following
components are included:
One gel casting tray with molded V-grooves for placement of gel casting dams
One gel deck
Two aluminum gel casting dams
Instruction manual on CD along with all Apogee Electrophoresis apparatus manuals
Due to the variety of applications and combs available, well-forming combs are not included. To form
sample wells, use the combs supplied with the H58 or purchased separately.

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2.0 GEL CASTING PROCEDURE
The procedure for casting a gel in the Horizon 58 Gel Casting System is fundamentally the same as that
used to cast gels in the H58 apparatus. For additional details on preparation of agarose gels and
formulations of common buffers refer to the H58 instruction manual.
1. Place the gel casting tray on a flat, level surface. Place the gel deck in the gel deck seat of the
casting tray between the two sets of V-grooves.
2. Slide the gel casting dams into the V-grooves. Apply even, gentle downward pressure to both
dams to seat the sealing surfaces of the dams against the ends of the gel deck. Do not force the
dams down –this can displace the deck out of level.
3. Place a small ‘bulls eye’level in the center of the deck to check that the surface of the gel deck
is level.
4. Insert a comb or combs (such as provided with the H58 apparatus) into the preferred alignment
slots of the casting tray. Ensure that each comb rests unobstructed and squarely in its slot and
on the raised edges of the gel deck. The system is now ready for gel casting.
Note: If you are using a 4-stripe gel deck or the 4-stripe side of a 3 and 4 stripe card, combs placed
in the center slot of the casting tray will not align precisely with the red well visualization stripes.
5. Prepare the desired volume of molten agarose in electrophoresis buffer in a bottle or
Erlenmeyer flask. For information on agarose preparation and buffer formulation see Section 4.
6. Loosely cap the container. Allow the molten agarose to cool to 50°C to 60°C.
Caution: Casting gels with agarose above 60°C will result in poor sealing at the gel casting dams and
may cause the bottom of the deck to bow.
7. Pour the measured volume of molten agarose into the center of the gel deck. Use a pipette tip
to distribute the agarose evenly over the surface of the gel deck and to remove any air bubbles,
particularly from around comb teeth.
Note: If molten agarose leaks from below gel casting dams, be sure dams are seated properly.
8. Allow the agarose to cool until thoroughly solidified, usually 15 to 30 min. Gently remove gel
casting dams and comb(s) before transferring the gel and gel deck to the buffer tray of the H58
Apparatus. Be sure that the gel deck is properly seated in the buffer tray and that the gel is level
before loading samples or starting electrophoresis.
9. To store gels prior to electrophoresis, remove the gel casting dams and combs, wet the gel
surface with a small amount of electrophoresis buffer and wrap the gel deck (with the gel still in
place) with plastic wrap or seal in a plastic bag. Store at 4°C. Securely sealed gels can be stored
at 4°C for up to one week.
10. After use thoroughly rinse the casting tray and dams and remove any residual agarose by rinsing
with deionized water. Wipe dry or allow to air dry before storing.

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3.0 TROUBLESHOOTING GUIDE
Some suggestions for resolving common gel problems are given below.
PROBLEM COMMENTS
Agarose solution leaks during casting. Verify that the sealing surfaces of the gel deck and
gel casting dams are clean.
Verify that the gel casting dams are properly seated.
Verify that the ends of the gel deck are flat and free
of nicks.
Cool the agarose to 50°C to 60°C before pouring.
Samples leak underneath the gel upon The bottom of the wells were torn when the comb was
loading. removed.
Pronounced ‘smiling’along one edge of the Gel was cast or electrophoresed out of level.
gel occurs (corresponding bands in different Use the ‘bull’s eye’ level to verify that the apparatus
lanes migrate slower toward one edge). is level prior to gel casting and electrophoresis.
‘Wiggly’or ‘slanting’bands (bands are not Verify that the wells are free of particles and bubbles
straight lines or parallel to the top edges of before and after loading samples.
the gel). 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.

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4.0 APPLICATIONS
4.1 CONSIDERATIONS FOR AGAROSE GEL ELECTROPHORESIS
4.1.1 SELECTING GEL CONCENTRATION
The choice of agarose concentration for a gel depends on the range of fragment sizes to be separated.
The typical agarose concentration is 0.3% to 2.0%. Large DNA fragments require low-percentage gels,
while small DNA fragments resolve best on high-percentage gels. Gels containing <0.5% agarose are
very weak and should be electrophoresed at a low temperature (~4°C). For routine electrophoresis,
0.75% to 1.0% agarose gels provide a wide range of separation (0.15 to 15 kb). Thin (2 to 3 mm thick)
and low-percentage agarose gels yield better photographs than thick or high-percentage gels, which
exhibit increased opaqueness and auto-fluorescence.
4.1.2 PREPARING AGAROSE FOR GELS
The following protocol yields a 1% (w/v) agarose gel. Varying the amount of agarose added in step 1 will
produce gels of higher or lower concentration. Determine whether Tris-acetate/EDTA (TAE) buffer or
Tris-borate/EDTA (TBE) buffer (formulas in tables 1 and 2) is preferable for your specific application. To
determine the volume of agarose solution required to produce gels of various thicknesses, see table 4.
Table 1. 10X TAE Electrophoresis Buffer
Component
Amount
Concentration
Tris base
48.4 g
400 mM
Na2EDTA•2H2O
7.4 g
20 mM
Sodium acetate, anhydrous
16.4 g
200 mM
Glacial acetic acid
17.0 ml
296 mM
Deionized water
to 1 L
-----
Note: This is a 10X concentration solution. Dilute with deionized water prior to use. Final pH should be
7.8 at 25°C.
Table 2. 10X TBE Electrophoresis Buffer
Component
Amount
Concentration
Tris base
121.1 g
1 M
Boric acid, anhydrous
55.6 g
0.9 M
Na2EDTA•2H2O
3.7 g
10 mM
Deionized water
to 1 L
-----
Note: This is a 10X concentration solution. Dilute with deionized water prior to use. Final pH should be
8.3 at 25°C.
1. Add 1 g of agarose per 100 ml of 1X TAE or 1X TBE electrophoresis buffer (see tables 1 and 2 for
buffer formulas) in a bottle or Erlenmeyer flask of at least twice the final volume of solution.
2. Loosely cap and weigh the flask.

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3. Dissolve the agarose in electrophoresis buffer by heating in a microwave oven or boiling water
bath with occasional mixing until no granules of agarose are visible.
4. Weigh the flask and adjust to the original weight with deionized water to compensate for
evaporation.
5. Put the capped flask in a water bath at 50°C to 60°C, and allow the agarose to equilibrate at that
temperature before pouring gels.
4.1.3 PREPARING SAMPLES AND LOADING THE GEL
The amount of DNA that can be loaded per well is variable and depends upon the number and size of
the DNA fragments and the cross-sectional area of the well (well width gel thickness). As a general
rule, the minimum amount of DNA detectable by ethidium bromide staining is 1 ng in a 5 mm wide band
on a 3 mm thick gel. For preparative purposes on a 3 mm thick gel, the amount of DNA loaded should
not exceed 50 ng per 5 mm wide band. Overloading a gel may cause trailing and distortion of bands.
Table 3 contains a formula for a sample loading buffer, which should be added to DNA samples prior to
loading. For alternative formulas for sample loading buffers, see References 1 and 2.
Table 3. 10X Sample Loading Buffer
Component
Amount
Concentration
Glycerol
5 ml
50% (v/v)
Na2EDTA•2H2O
0.37 g
100 mM
Sodium dodecyl sulfate
0.1 g
1% (w/v)
Bromophenol blue
0.01 g
0.1% (w/v)
Deionized water
to 10 ml
------
Note: This is a 10X concentration solution. Add 0.1 volume of buffer to samples and apply directly to
gel. If the samples contain l-cohesive ends, as with l DNA restriction fragments, the samples in buffer
should be heated at 65°C for 5 to 10 min prior to loading.
The sample volumes that can be loaded per well for each standard H58 Apparatus comb are listed in
table 4. For analytical purposes, keep sample volumes to a minimum. Generally, 0.8 mm thick combs
provide sharper band definition than 1.5 mm thick combs.

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Table 4. Sample Volumes for Horizon 58 Combs as a Function of Gel Thickness
Gel Thickness
Agarose Volume
(ml)
Comb Thickness
(mm)
Number of Wells
Capacity/Well (ul)
3 Well
15
0.8
5
15
8
7
14
3
1.5
3 (a)
140
5
30
8
15
14
7
4 Well
20
0.8
5
20
8
10
14
5
1.5
3 (a)
200
5
40
8
20
12 (b)
20
14
10
5 Well
25
0.8
5
30
8
15
14
7
1.5
3 (a)
250
5
50
8
25
14
13
a. Includes one preparative well and two flanking reference analytical wells for standards
(dimensions and capacity values are for the central preparative well).
b. 4.5 mm spacing
Note: Volumes given are approximate. Low-percentage gels (<0.6%) and low-melting-point agarose gels
may have lower sample well volumes.

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4.2 ETHIDIUM BROMIDE STAINING OF DOUBLE-STRANDED DNA
To visualize double-stranded DNA after electrophoresis, the gel should be transferred from the deck to a
0.5 µg/ml solution of ethidium bromide in deionized water. Approximate staining time is 10 to 15 min
for a 3 mm thick gel and longer for thicker gels. As an optional subsequent step to reduce background
fluorescence, the gel can be destained in deionized water for 15 to 30 min.
Alternatively, ethidium bromide may be added directly to the agarose prior to casting, so that the gel is
electrophoresed in the presence of ethidium bromide. However, this procedure reduces the migration
rate and may alter the relative electrophoretic mobility of nucleic acids (reference 3).
4.3 GEL PHOTOGRAPHY
A darkroom or light-tight enclosure, camera, digital camera and UV light source are required for
photography of gels stained with ethidium bromide. For best results, place the stained gel directly on
top of a 300 nm or 254 nm transilluminator. If the camera contains ASA 3000 or equivalent film the
required exposure at maximum aperture (f/4.5) should be between 1/4 and 2 seconds. Intensity of the
light source, distance between the gel and the camera lens, film or shutter speed, lens aperture, and
choice of photographic filters will all affect exposure time.
Transmitted UV light yields the highest sensitivities (1 ng of DNA in a 5 mm wide band) in photographing
gels. Photography under incident UV light is approximately 10 times less sensitive. A UV-blocking
filter (Kodak 2B Wratten filter) used in conjunction with a red gelatin filter (Kodak 23A Wratten filter)
provides the highest contrast. Due to the fluorescence of the 2B filter, the two filters must be oriented
so that the red 23A filter is adjacent to the camera lens. The ethidium bromide-DNA complex fluoresces
at 590 nm upon excitation at 302 nm. Short-wave (254 nm) sources provide an equivalent level of
sensitivity; however, high-energy UV causes photodimerization and nicking of the DNA. Long-wave
transilluminators (366 nm) are much less efficient.
REFERENCES
1. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York.
2. Rickwood, D. and Hames, B.D. (eds.) (1982) Gel Electrophoresis of Nucleic Acids: A Practical
Approach, IRL Press, Oxford, England.
3. Longo, M.C. and Hartley, J.L. (1986) LTI FOCUS® 8:3.

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5.0 RELATED PRODUCTS AND REPLACEMENT PARTS
5.1 H58 ACCESSORIES DESCRIPTION CATALOG #
Preparative Delrin Comb with marker lanes 1.5 mm thick 21065131
5 well precision machined white Delrin comb 0.8 mm thick 21065099
1.5 mm thick 21065107
8 well precision machined white Delrin comb 0.8 mm thick 21065073
1.5mm thick 21065081
14 well precision machined white Delrin comb 0.8 mm thick 21065115
1.5 mm thick 21065123
12 well for use with multichannel pipettes (4.5 mm spacing) 1.5 mm thick 11951142
Gel Deck (shipped without visualization stripes) Each 21065164
H58 Aluminum Casting Dams Package of 2 21065032