C.B.S. SCIENTIFIC DGGE-1001 User manual

Denaturing Gradient Gel Electrophoresis Systems
INSTRUCTION MANUAL
DGGE-1001 DGGEK-1001
DGGE-2001 DGGEK-2001
DGGE-2401 DGGEK-2401
DGGE-4001 DGGEK-4001
DGGE-4801 DGGEK-4801
shown above: DGGEK-2401

TABLE OF CONTENTS
Page
Important User Information 3-4
Section 1 General Information
1.1 Introduction 5
1.2 Specifications 5
1.3Safety 6
Section 2 Description of parts
2.1 Unpacking 7
2.2 Components/Assembly 7
Section 3 Instructions for Use
3.1 Unit Set-up and Unpacking 8-11
3.2 Preparation/Cleaning of Glass Plates for Gel Casting 12
3.3 Gel Casting Techniques 12-16
3.4 Vertical Gradient Formation 17
3.5 Preparation of the Cassettes 18
3.6 Buffer Cycling Connections 19-20
3.7 Running the Gels 21
3.8 Removing the Gels 22
3.9 Perpeniduclar Gel Casting 23
3.10 Perpendicular Gradient Formation 24-25
3.11 Buffer Siphon Pump Instructions for use 26
3.12 Maximum Well/Comb Volumes 27
Section 4 Running Conditions
4.1 Recommended Power 27
4.2 Recommended Buffers 27-28
4.3 References 28
Section 5 Maintenance of Equipment
5.1 Care and Handling 29
5.2 Maintenance 29
Section 6 DGGE System and Accessories 30-31

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IMPORTANT USER INFORMATION
This Instruction Manual will explain how to use this product safely and effectively. Please read and
carefully follow the instruction manual in its entirety.
The triangle/exclamation mark symbol alerts the user of the product to important
operational, maintenance, and/or warranty requirements.
The triangle/lighting bolt symbol alerts the user of the product to potentially hazardous
electrical exposure.
Failure to adhere to the instructions could result in personal and/or laboratory hazards, as well as
invalidate any warranty. Always turn off the DC power source prior to disconnecting power cords
from the product. Disconnect power cords from the power source first, and then from the
product. For maximum safety, always operate this system in an isolated, low traffic area, not
accessible to unauthorized personnel. Never operate damaged or leaking equipment.
WARRANTY AND LIABILITY
This product was produced utilizing the highest practical standards of materials, workmanship, and
design. C.B.S. Scientific warrants that the product has been tested and will meet or exceed published
specifications. This warranty is valid only if the product has been operated and maintained according to
the instructions provided.
C.B.S. Scientific warrants this product to be free from defects in materials and workmanship under normal
service for one year from date of shipment. If the product proves defective during this period, C.B.S.
Scientific will repair or replace it at our option, free of charge, if returned to us postage prepaid. This
warranty does not cover: damage in transit, damage caused by carelessness, misuse or neglect, normal
wear through frequent use, damage caused by solvent corrosion, damage caused by improper handling
or user alteration, nor unsatisfactory performance as a result of conditions beyond our control. C.B.S.
Scientific shall in no event be liable for incidental nor consequential damages, including without limitation,
lost profits, loss of income, loss of business opportunities, loss of use and other related damages,
however caused, nor any damage arising from the incorrect use of the product.
EN
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FRANÇAIS INFORMATION IMPORTANTE À L'USAGE DES UTILISATEURS
Le présent manuel d'utilisation explique la manière de se servir efficacement du produit en conditions
de sécurité. Il est recommandé de soigneusement lire la totalité du manuel, avec ses consignes
et ses instructions.
Le triangle avec point d'exclamation est un symbole destiné à avertir l'utilisateur du produit de
l'importance de certaines exigences relatives au fonctionnement, à l'entretien et/ou à la
garantie.
Le triangle avec flèche en zigzag est un symbole destiné à avertir l'utilisateur du produit de la
possibilité d'exposition à des décharges avec danger de secousses électriques.
Tout manquement à l'observation des consignes et des instructions peut exposer les personnes
et les biens à des dommages corporels et/ou matériels et peut annuler toute garantie. Il faut toujours
interrompre l'alimentation de courant continu avant de déconnecter les cordons d'alimentation du
produit. Déconnecter d'abord les cordons d'alimentation branchés sur la source de tension
(alimentation de secteur) puis ceux branchés sur le produit. Pour une sécurité maximum, il faut toujours
faire fonctionner ce système dans un lieu isolé, peu fréquenté, où le personnel non autorisé n'a pas
accès. Ne jamais faire fonctionner un matériel endommagé ou affecté par des fuites.
GARANTIE ET RESPONSABILITÉ
Le produit a été fabriqué conformément aux normes applicables les plus exigentes en matière de
matériaux, de main d'oeuvre, de conception et d'ingéniérie. C.B.S. Scientific garantit que le produit a
subi des essais et que ses performances rempliront les conditions des spécifications publiées ou leur
seront même supérieures. La présente garantie n'est valide que si le produit a fonctionné et a été
entretenu conformément aux consignes et instructions fournies.
C.B.S. Scientific garantit que le produit sera dépourvu de vices de matériaux et de main d'oeuvre, en
conditions de service normales, pendant un an à compter de la date d'expédition. Au cas où le produit
s'avérerait défectueux pendant cette période de garantie, C.B.S. Scientific réparera ou remplacera le
produit, à sa discrétion et gratuitement, si le produit lui est retourné port payé d'avance. La garantie ne
couvre pas les dommages de transport; les dommages causés par l'imprudence, le manque de soins,
l'abus ou la négligence; l'usure normale résultant d'une utilisation fréquente; les dommages causés par
la corrosion des solvants; et les dommages causés par la manipulation inadéquate ou des
changements apportés par l'utilisateur. La garantie ne couvre pas non plus les performances non
satisfaisantes résultant de conditions hors du contrôle de C.B.S. Scientific. C.B.S. Scientific ne pourra
en aucun cas être tenue responsable de dommages indirects, y compris, de manière non limitative, la
perte de bénéfices, le manque à gagner, la perte d'occasions d'affaires, l'impossibilité d'usage ou tous
autres dommages associés, quelle qu'en soit la cause, ni de dommages résultant de l'usage incorrect
du produit.
ESPAÑOL INFORMACIÓN IMPORTANTE PARA EL USUARIO
El presente instructivo explica la manera de usar este producto en forma segura y efectiva. Sírvase
leerlo en su totalidad y seguir detenidamente las indicaciones que contiene.
El símbolo del triángulo con exclamación llama la atención del usuario a requisitos
importantes para el uso y mantenimiento del producto, así como para la validez de la
garantía.
El símbolo del triángulo con rayo llama la atención del usuario a la posibilidad de
riesgos eléctricos.
El incumplimiento de las instrucciones aquí señaladas podría dar lugar a riesgos a la persona, al
laboratorio o a ambos y podría anular toda garantía. Siempre apague la fuente de corriente continua
antes de desenchufar los cables eléctricos del producto. Primero desconecte los cables de la fuente
de energía y después del producto. Para mayor seguridad, siempre use este sistema en un área
aislada, de poco movimiento de personas e inaccesible a personal no autorizado. Jamás use equipo
que presenta algún daño o fuga.
GARANTÍA Y RESPONSABILIDAD
Este producto fue fabricado de acuerdo con las normas más estrictas que sean factibles en cuanto a
materiales, mano de obra y diseño. C.B.S Scientific garantiza que se sometió el producto a pruebas y
que cumplirá o excederá las especificaciones publicadas. Esta garantía será válida únicamente si se
usa y se da servicio de mantenimiento al producto de acuerdo con las instrucciones señaladas.
C.B.S. Scientific garantiza que este producto se encontrará libre de defectos de materiales y mano de
obra por un período de servicio normal de un año a partir de la fecha de embarque. Si el producto
resulta defectuoso durante este período, C.B.S. Scientific lo reparará o lo repondrá, a criterio de C.B.S.,
libre de cargos, si se devuelve el producto a C.B.S. porte pagado. Esta garantía no cubre daños
sufridos en tránsito, daños provocados por descuido, mal uso o negligencia, desgaste normal como
consecuencia del uso excesivo, daños atribuibles a corrosión provocada por solventes, daños
causados por el uso indebido o alteraciones realizadas por el usuario ni rendimiento insatisfactorio
atribuible a circunstancias fuera del control de C.B.S. Scientific. C.B.S. Scientific en ningún caso
asumirá responsabilidad por daños incidentales o subsecuentes, incluyendo, en forma no limitativa, la
pérdida de utilidades, de ingresos, de oportunidades comerciales o del uso del producto y otros daños
afines, fuere cual fuere su origen, ni por daños derivados del uso incorrecto del producto.
DEUTSCH WICHTIGE INFORMATION FÜR DEN BENUTZER
Diese Bedienungsanleitung beschreibt wie man dieses Produkt sicher und wirksam benutzt. Bitte lesen
und befolgen Sie alle Anweisungen in dieser Anleitung.
Das Dreieck mit Ausrufezeichen weist den Benutzer des Produktes darauf hin, daß
wichtige Bedienungs-, Wartungs- und/oder Garantievorschriften zu beachten sind.
Das Dreieck mit Zickzackblitz warnt den Benutzer des Produktes vor möglichen
Gefahren durch elektrische Spannungen.
Nichtbeachtung dieser Anweisungen kann zu persönlichen und/oder labortechnischen Schäden
führen und gleichzeitig alle Garantien als nichtig erklären. Die DC Stromzufuhr muß immer, vor dem
Entfernen der Stromkabel vom Produkt, abgeschaltet werden. Die Stromzufuhrkabel müssen zuerst
von der Steckdose und erst dann vom Produkt entfernt werden. Um höchste Sicherheit zu
gewährleisten sollte dieses System in einem abgesonderten und besonders ruhigen Bereich eingesetzt
werden und vor Unbefugten sicher sein.
GARANTIE UND HAFTUNG
Dieses Produkt wurde unter Anwendung von Produkten mit höchster Qualität und aus Materialien mit
bester Verarbeitung und modernstem Design hergestellt. C.B.S Scientific garantiert, daß das Produkt
getestet wurde und alle publizierten Spezifikationen übertrifft. Diese Garantie ist jedoch nur gültig,
wenn das Produkt nach der beigefügten Bedienungsanleitung bedient und gewartet wurde.
C.B.S. Scientific garantiert, daß dieses Produkt bei normaler Bedienung aus fehlerfreiem Material
besteht und fehlerfrei in der Ausführung ist. Diese Garantie gilt für ein Jahr ab Lieferdatum. Sollte das
Produkt in diesem Zeiraum fehlerhaft werden, bietet C.B.S. Scientific eine kostenlose Reparatur bzw.
Kostenlosen Ersatz, einschließlich freiem Rückporto. Diese Garantie schließt folgendes aus:
Transportschaden, Schaden durch Nachlässigkeit, Mißbrauch oder Vernachlässigung, normale
Abnützung durch regelmäßigen Gebrauch, Schaden durch Säureangriff, Schaden durch falsche
Handhabung, Veränderung des Produktes durch den Benutzer, oder unzureichende Leistungen die sich
nicht im Verantwortungsbereich von C.B.S. Scientific befinden. C.B.S. Scientific kommt unter keinen
Umständen für folgende Schäden auf: Sachschadensverlust, Einkommensverlust, Verlust von
Geschäftsmöglichkeiten, Verlust der Anwendung und andere damit verbundene Schäden die auf irgend
eine Art und Weise entstanden sind, oder Schäden die aus falscher Anwendung des Produktes
entstanden sind.
ITALIANO INFORMAZIONI IMPORTANTI PER L’UTENTE
Questo manuale spiega come utilizzare questo prodotto in maniera sicura ed efficiente. Si pregai di
leggere e seguire con cautela le istruzioni di ogni parte di questo manuale.
Il triangolo contenete il simbolo di un punto esclamativo avverte l’utente di importanti
requisiti relativi al funzionamento, manutenzione e/o garanzia del prodotto.
Il triangolo contenete il simbolo di un lampo avverte l’utente del prodotto della
possibilità di pericoli dovuti a corrente elettrica.
La mancata osservanza delle istruzioni può essere causa di pericolo alla propria persona ed al
laboratorio, oltre a poter annullare la garanzia. Prima di distaccare il cordone d’alimentazione dal
prodotto, spegnere sempre la sorgente di corrente continua. Distaccare i cordoni d’alimentazione
prima dal lato della sorgente di tensione e poi dal lato del prodotto. Per maggior sicurezza, mettere
sempre in funzione il prodotto in un’area isolata con poco traffico che non sia accessibile al personale
non autorizzato. Non mettere mai in funzione un’apparecchiatura che sia danneggiata o abbia perdite.
GARANZIA E RESPONSABILITÀ
Questo prodotto è stato fabbricato seguendo gli standard più elevati per i materiali, la manodopera e la
progettazione. La C.B.S. Scientific garantisce il prodotto è stato sottoposto a prova e raggiunge o
supera i valori pubblicati per i dati tecnici. Questa garanzia è valida solo se il prodotto è messo in
esercizio e soggetto a manutenzione secondo le istruzioni fornite.
La C.B.S. Scientific garantisce che questo prodotto è libero di difetti di materiali e manodopera, in
normali condizioni d’esercizio, per la durata di un anno dalla data di spedizione. Se, in questo periodo, il
prodotto si dimostrerà difettoso, la C.B.S. Scientific, a suo giudizio, lo riparerà o sostituirà. Questa
garanzia non copre danni in transito, danni causati da negligenza, uso improprio, trascuratezza,
normale consumo derivante da uso frequente, o danni causati da solventi corrosivi, danni causati da
maltrattamento o da modifiche apportate dall’utente e non copre prestazioni insoddisfacenti che siano il
risultato di condizioni al di fuori del controllo del fabbricante. La C.B.S. Scientific non sarà in ogni caso
responsabile per danni incidentali o consequenziali, incluso, senza limitazioni, perdite di profitto, perdita
di entrate, perdita di opportunità d’affari e altri danni relativi, comunque causati, e per danni risultati da
uso incorretto del prodotto.

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SECTION 1
General Information
1.1 Introduction
Denaturing Gradient Gel Electrophoresis (DGGE) is a powerful genetic analysis technique that can be used
for detecting single base changes and polymorphisms in genomic (1, 2), cloned, and PCR amplified DNA
(3,4). Two of the most valuable uses for DGGE in human genetics are in directly detecting single base
changes that cause disease and in detecting polymorphisms with DNA probes for genetic-linkage analysis.
Also, DNA fragment melting points can be determined using Perpendicular DGGE (1).
C.B.S. Scientific has designed five different DGGE Systems, which are reliable and easy-to-use. The
DGGE-1001 is a 2gel system and features one dual gel cassette. The DGGE-2001 features two single gel
cassettes. The DGGE-2401 has the same buffer tank as the DGGE-1001 and DGGE-2001 but features two
dual gel cassettes, for a capacity of 4 gels per run. Also available is the DGGE-4001, which has a larger
buffer tank and four single cassettes. Finally there is the DGGE-4801, which features the larger buffer tank
and four dual cassettes enabling the researcher to run up to 8 gels simultaneously. Improvements to the
technique include: a simplified method for casting perpendicular and vertical gels using Gel Wrap, single or
dual gel cassettes have been redesigned eliminating the use of agarose plugs, the need for an external
peristaltic pump for buffer cycling has been eliminated, bulky screw clamps have been replaced with
polypropylene spring clamps, buffer tank dimensions have been changed to use bench space more
efficiently, and a safety cover with an electrical interlock has been added for protection and to help maintain
temperature and reduce evaporation.
1.2 Specifications
Constructions
DGGETank Acrylic
DGGELid Sodalime glass, acrylic
Electrodes Platinum wire .010" diameter
Power cords FR Silicone, rated 7500V,
200mA, 65C
DGGE Cassette Acrylic
Heater/Stirrer Buffer Cycler Stainless steel
Combs Teflon
Spacers Polymer
Glass Plates Sodalime or Borosilicate
Gradient Maker Acrylic, teflon, stainless steel
DGGE-
1001 DGGE- 2001 DGGE- 2401 DGGE- 4001 DGGE- 4801
Shipping weight 40lbs 58lbs 58lbs 68lbs 108lbs
Gel Size 15x20cm 15x20cm 15x20cm 15x20cm 15x20cm
Buffer Volume 15.2 L** 21 L 21L
Distance between
electrodes
20cm 20cm 20cm 20cm 20cm
Voltage limit 250 250 250 250 250
**requires the use of a buffer displacement chamber

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1.3 Safety
Power to the DGGE systems is to be supplied by an external DC voltage power supply that must be
ground isolated so that the DC voltage output floats with respect to ground. For any power supply used,
the maximum specified operating parameters for the units are:
Maximum Limits
250 VDC voltage
30 watts power
80 mA current
70C ambient temperature
Current to the unit, provided from the external power supply, must enter the unit through the lid
assembly, providing a safety interlock to the user. DC current to the unit is broken when the lid is
opened. Do not attempt to use the unit without the safety lid. Always turn the power supply off
before removing the lid, or when working with the unit in any way. Follow safety precautions
specified by the power supply manufacturer.
Input Power: Mains to safety interlock
Depending on country of destination, input voltage from mains electrical supply are as follows: 110-
120VAC @ 50/60Hz/5 Amps or 200-240VAC @ 50/60Hz/5 Amps. Country specific power cords or CE
approved adapter kits are supplied with each system.

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SECTION 2
Description of Parts
2.1 Unpacking
Please verify that your DGGE or DGGEK system comes complete with the following components:
Lower Reservoir/Glass tank/safety interlock/ with 2 black leads inside lid
Heater/Stirrer/By-pass pump with clear plastic vapor shield
Gel cassette(s) as specified (single or dual, single cassettes shown in Figure 2.1)
1 Thermometer
1 Gradient maker, 20mls per side, Cat. # GM-40
2 each combs, 2 sets glass plates, 2 pair spacers and 2 each Gel Wrapgaskets
White spring clamps, 12 each GPC-0002-177 and 16 each GPC-0001-177 (Number of clamps
received with system will vary depending on format of DGGE)
Buffer siphon pump with tubing, Cat# BSP-1000
DGGEK Series also includes:
EPS-300 or EPS-300-II Power supply
Peristaltic Mini-Pump for gel casting
2.2 Components/Assembly
Heater
Stirrer
Buffer Siphon
PumpAssembly
Vapor
Shield
Thermomete
r
DGGE
Tank
Single
Cassetes
Comb (2)
Spacers
(2 pairs)
Glass Plate
Assembly(2)
Clamps
Mini-Pump
(Optional)
GM-40
Gelwrap
® (2)

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SECTION 3
Instructions for Use
3.1 Unit Set-up and Unpacking Instructions
1. Unpack lower reservoir and place on level surface in an approved location
2. Install Heater/Stirrer as shown below. Secure in place with thumbscrew. Be certain that the clear plastic vapor
shield is between the bottom side of the heater control unit and the TOP surface of the tank lid. Small bore
silicone tubing for buffer cycling is already attached to the manifold outlets. Connect large bore tubing from
bypass pump to barb fitting on plastic manifold on underside of lid. WARNING: Do NOT turn on
Heater/Stirrer until tank has been filled with buffer.
Heater/Stirrer/Bypass Pump Installation
Mountin
g
Bracket
Clear Plastic
Vapor Shield
Bypass Pump
(
Buffer Recirculation
)
Buffer C
y
clin
g
Manifold
Fig. 3-1

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3.1 Unit Set-up and Unpacking Instructions-continued
3. Install thermometer into red silicone stopper on lid. Moisten before inserting. See Figure below.
Fig. 3-2

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3.1 Unit Set-up and Unpacking Instructions-continued
4. Position an appropriate power supply on a shelf above the tank.
5. Fill the tank with running buffer (see chart page 5) “ To obtain the correct level of buffer, place the
cassettes in the tank and fill the tank until the level of buffer reaches the underside of the upper
reservoir. The upper reservoir holds only 50mls.
6. Attach the power supply to the safety interlock. Refer to Figure below.
7. Attach the safety interlock to a wall (mains) outlet.
8. Plug the Heater/Stirrer into a wall (mains) outlet.
9. Do NOT attach power leads (from power supply to safety interlock) until gels are loaded and
installed in the tank.
Power Supply/Safety Interlock Connections
To mains
(wall outlet)
To mains
(wall outlet)
Fig. 3-3

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3.1 Unit Set-up and Unpacking Instructions-continued
10. Turn Heater/Stirrer “On” and set temperature to 60C (Figure 3.2)
Fig. 3-4
Temperature Setting 60C

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3.2 Preparation/Cleaning of Glass Plate for Gel Casting
Hand wash both plates with a high quality lab detergent followed by a complete rinsing with
dH2O. Air dry or use a lint-free tissue. Spray/wipe the chosen inner surfaces of the plate set
with 95% ethanol and dry with lint-free tissue.
3.3 Gel Casting Techniques.
A. Gel Wrap™ Gasket Casting Method.
B. Vertical gradient gel casting using GM-40 gradient maker and gravity flow.
C. Vertical gradient gel casting using GM-40 gradient maker and a Mini-pump.
D. Vertical gradient gel casting using GM-40 gradient maker, Mini-pump and Multi-gel
Caster.
NOTE: Prior to casting, mark the notched glass plate to designate which side is the ‘inside’ and
‘outside’. The same side of the notched plate should always sit against the reservoir gasket.
A. Gel Casting using Gel WrapGasket Casting method
For Vertical DGGE, use the set of spacers which do not have the small hole or channel milled
into the lower end, these are for casting perpendicular gels.
1. Start by holding the rectangular back
plate with the rounded bottom corners
and start applying the gasket around
one side of the glass plate. Note: one
side of the “U” shaped gasket is flat,
and the other side has tubing that will
act as a seal around the spacers.
2. When applying the gasket over the
rounded corners of the back glass plate,
make sure the notches on the gasket
align with the rounded corners of the
glass plate. Once the gasket is pushed
over the bottom edge and corners, work
it down the remaining side.

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3. Place the gasketed plate on the lab bench
with the tubing side up, and extend the
bottom of the plate over the edge of the
bench, approximately ¾ of an inch. Place
the spacers along side the inside edges
of the gasket. Be sure the rounded corner
end of each spacer is facing the outside
bottom of the plate, following the radius
of the glass.
3.3 Gel Casting Techniques.
A. Gel WrapGasket Casting method-continued
4. Place the notched plate on top of the
bottom assembly, starting from the bottom
edge and gently easing the plate down.
Verify the gasket is smooth around the
edges and then clamp along the bottom.
5. Lift the assembly and stand it on the base
of the clamps. For leveling, push glass
plate assembly down until it stops against
clamp body. Clamp the sides of the
assembly with additional casting clamps
on either side. As each clamp is attached,
be sure the gasket is aligned between the
plates forming a seal.

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3.3 Gel Casting Techniques- continued
B. Vertical gradient gel casting using GM-40 gradient maker and gravity flow.
1. Place the GM-40, gradient maker, on an elevated magnetic stirrer with a small “flea” stir
bar in the cylinder (C-2) closest to the outlet. The gradient maker should be fitted with a
leur valve, (V-2) and a 20ga needle with attached tubing to deliver acrylamide to the gel
plate sandwich as shown in figure (3.5). With the height differential between the leur valve
(V-2) and the top of the gel plate sandwich between 2-4 inches, it takes approximately 5-9
minutes to cast the gel. Raising the height of the gradient maker can increase flow rate.
Flow can be regulated by turning leur valve (V-2). Flow rate appropriate for each gradient
should be determined empirically.
C
-1 C-2
V-1 V-2
Fig. 3-5

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3.3 Gel Casting Techniques- continued
C. Vertical gradient gel casting using using gradient maker (GM-40) and a mini-pump.
1. Alternatively, you may choose to use a “mini-pump” or other peristaltic pump to cast
gels as shown in fig (3.6). If so, secure the gradient maker to a ring stand and connect
the outlet tubing to the mini-pump tubing adapter. Connect tubing from mini-pump to a
20ga. needle for affixing between glass plates.
Fig. 3-6

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D. Vertical gradient gel casting using GM-40 gradient maker, Mini-Pump and Multi-Gel
Caster.
1. Clean the gel caster with soap and water and the glass plates with alcohol.
2. Place a separating sheet in the gel caster.
3. Place the back plate (non-eared) on the sheet.
4. Place the 0.75mm spacers on each side of the back plate.
5. Place the top plate (eared) on the spacers to form 1st sandwich. Repeat as required.
Continue to Section 3.4 Vertical Gradient Formation.
Mini-Pump
Magnetic Stirrer
Gradient Maker Multi-Gel Caster
Stopcock
SC-2001
Silicone Tubing
SCT-100
Fig. 3-7

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3.4 Vertical Gradient Formation
To determine the range of gradient appropriate for your fragment analysis, please read
the enclosed paper by Myers, Sheffield and Cox, especially section 6.1.3 through 6.2.1
(pages 124 to 126). This gives you an excellent overview of the determination of
melting behavior of your fragments.
1. The following is a typical protocol for casting a 40%-60% gradient gel. Refer to Section
3.4 Vertical Gradient Formation for apparatus assembly. In an ice bucket, place two
50ml conical tubes labeled “A” and “B”. Add to tube “A”
11.5ml of 40%/7.5%
80 I (10%) APS
5 l TEMED
2. Add to tube "B":
11.5ml of 60%/7.5%
80 I (10%) APS
5 I TEMED
3. Pour solution "B" into right side of gradient maker (C-2),(GM-40) (fig. 3.5), and
open interior valve to allow air bubble to escape. Let as much as 1 ml "B" solution
BACKFLOW into left side of gradient maker. Decant the 1 ml back into right side with
pasteur pipette. Remove any residual solution from (C-1) with absorbent paper.
4. Add solution "A" to left side of gradient maker.
5. Turn on magnetic stirrer.
6. Exit tube with needle should be secured to gel plate notch with tape.
7. Open inside valve first (V-1), then outside (V-2) valve to start gravity flow.
Optionally, Turn on mini-pump and pump acrylamide at medium speed. Determine
optimal speed empirically.
8. Gel volume is 23ml, using the 0.75mm spacers. If gel volume is not enough to fill
sandwich, use 0% to "top-off". If using water saturated butanol for overlay, leave 0.5cm
void to create flat interface.
9. Insert the 16-well rectangular tooth comb. Allow the gel to polymerize for 20-30
minutes.

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3.5 Preparation of the Cassettes
1. After polymerization rinse gel plate assembly with D.I. water to remove excess
acrylamide or denaturants from plate exterior.
2. Remove comb and quickly transfer gel sandwich to cassette by removing all #2
clamps. LEAVE GEL WRAP IN PLACE. The Gel Wrap acts as a barrier and prevents
perpendicular electrical fields from intenferring with outside lanes. Re-clamp to
cassette using #1 clamps/4 per side. Dislodge by pulling the bottom section of the Gel
Wrap from the plates to allow buffer contact with the gel. See figure 3.8.
3. Immerse cassette into pre-heated buffer tank (fig.3.8), and attach recirculating tubing to
each upper reservoir (fig. 3.9 a,b,c). Rinse loading interface of gel with tank buffer to
remove non-polymerized acrylamide or excess urea. Rinse each well with buffer and fill
upper reservoir
Dislod
g
e Gelwra
p
Fig. 3-8

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3.6 Buffer Cycling Connections
Buffer Cycling Connections - Single
Fig. 3-9a
Buffer Cycling Connections – Two Single Cassettes

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Fig. 3-9b
3.6 Buffer Cycling Connections-continued.
Fig. 3-9c
1. Attach small bore tubing to barb fitting on cassettes.
2. Turn heater/stirrer on to fill upper reservoir; begin buffer cycling.
Buffer Cycling Connections-
Dual Cassette