Amersham Biosciences Hoefer DALT User manual

80-6431-50 Rev A/12-98
Hoefer DALT System
DALT Electrophoresis Tank
DALT Gradient Maker
DALT Multiple Gel Caster
DALT Blotting Kit
User Manual

AmershamBiosciences
Important User Information
Please read this entire manual to fully understand the safe
and effective use of this product.
The exclamation mark within an equilateral triangle is intended to alert the
user to the presence of important operating and maintenance instructions in
the literature accompanying the instrument.
If you have any comments on this manual, please send them to us at:
Amersham Biosciences Inc.
Marketing Department
654 Minnesota Street
San Francisco, CA 94107 USA
AmershamBiosciences reservesthe righttomake changesinthe specifications
without priornotice.
Warranty and Liability
Amersham Biosciences guaranteesthat the product deliveredhasbeen thoroughly
testedto assurethat it meetsitspublished specifications.Thewarranty included inthe
conditions of delivery is valid only if the product has been installed and used according to
theinstructionssuppliedby Amersham Biosciences.
Amersham Biosciences shall in noevent beliableforincidental orconsequential
damages,including without limitation,lostprofits, loss ofincome,lossofbusinessoppor-
tunities, loss of use and other related exposures, however caused, arising from the faulty
and incorrect use of the product.
©1998Amersham Biosciences Limited.
All rights reserved.
No part of this publication may be reproduced, stored in a retrieval system or transmitted
in any form by any means, without permission in written form from the company.
English

Hoefer DALT System
Amersham Biosciences
Renseignements importants
d’utilization
Pour une bonne compréhension et une utilisation
en sécurité maximale, il convient de lire entièrement ce manuel.
Dans la documentation qui accompagne l’instrument un point d’excla-
mation dans un triangle équilatéral a pour but d’attirer l’attention de
l’utilisateur sur des instructions importantes de fonctionnment ou de
maintenance.
Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser
à:
Amersham Biosciences Inc.
Marketing Department
654 Minnesota Street
San Francisco, CA 94107 USA
Amersham Biosciences seréserveledroitd’effectuerdesmodificationsde
ces spécifications sans aucun préavis.
Garantieet responsabilité
Amersham Biosciences garantit àl’utilisateurqueleproduitlivréa subi
avec succèstouslesessaisprévuspours’assurerqu’il estconforme aux spécifica-
tionsetnormesenvigueur. Lagarantieinclusedanslesconditionsdelivraison
n’est valablequesi leproduit a étéinstalléetutiliséconformément aux instruc-
tions fournies parAmershamBiosciences.
Lasociété AmershamBiosciences nesera enaucuncasresponsable de
tout dommagecausédirectementouindirectementpartouteutilisation incor-
recteou nonapprouvée du produit oudécoulant decetteutilisation, y compris
toute perte de bénéfice ou de recettes, toute perte de perspectives commerciales,
tout empêchement d’utilisation ettoutautrerisquesayantunrapportavec l’util-
isation du produit, mais sans aucune limitation quant à la nature de ces
dommages.
©1998AmershamBiosciences Limited.
Tous droits réservés.
La reproduction, le stockage dans un système de récupération d’informations ou
la transmission sous quelque forme que ce soit et par quelque moyen que ce soit
de la présente publication en totalité ou en partie sont strictement interdits sans
autorisation préalable écrite de la société.
Información importante para el usuario
Para comprender el producto y utilizarlo con segu-
ridad es necesario leer este manual en su totalidad.
El signo de admiración en un triángulo
equilátero en el manual, advierte al usuario sobre la presencia de
instrucciones importantes de operación y mantenimiento del aparato.
Si desearan hacer algún comentario sobre este manual, tengan la amabilidad de
remitirlo a:
Amersham Biosciences Inc.
Marketing Department
654 Minnesota Street
San Francisco, CA 94107 USA
Amersham Biosciences sereserva el derecho amodificarlasespecifica-
ciones sin previo aviso.
Garantía y responsabilidad
Amersham Biosciences garantizaqueel productoentregado hasidopro-
badoafondo paracomprobarelcumplimientodelas especificacionespublica-
das. La garantía incluida en las condiciones de entrega sólo es válida si el
producto se ha instalado y utilizado de acuerdo con las instrucciones entregadas
porAmersham Biosciences.
Amersham Biosciences noseráresponsable, bajo ningún concepto, de
daños directos o indirectos, incluyendo sin limitación la pérdida de beneficios, la
pérdida de ingresos, la pérdida de oportunidades de negocio, la pérdida de uti-
lización y otras consecuencias relacionadas, cualquiera que sea la causa, que se
deban a lautilización defectuosaeincorrecta del producto.
©1998Amersham Biosciences Limited.Reservadostodos
los derechos.
No está permitida la reproducción, ni el almacenaje en un sistema de recuper-
ación, ni la transmisión de parte alguna de esta publicación sin la autorización
por escrito de la empresa.
Wichtige Benutzerinformationen
Für ein vollständiges Verständnis und eine sichere
Handhabung dieses Produktes ist es notwendig, daß
der Benutzer dieses Handbuch vollständig durchliest.
Ein Ausrufezeichen in einem gleichseitigen Dreieck soll den
Benutzer auf die Anwesenheit wichtiger Betriebs- und Wartungsan-
weisungen in der dem Gerät beiliegenden Dokumentation hin-
weisen.
Wenn Sie Anmerkungen zu diesem Handbuch haben, dann senden Sie diese bitte
an:
Amersham Biosciences Inc.
Marketing Department
654 Minnesota Street
San Francisco, CA94107 USA
AmershamBiosciences behältsichdas Rechtvor,die Spezifikationen
ohne vorhergehende Ankündigung zu ändern.
Gewährleistung and Haftung
Amersham Biosciences garantiert, daß dasgelieferte Produkt sorgfältig
aufdieEinhaltungderveröffentlichtenSpezifikationen getestetwurde.Diein
den Lieferbedingungen näher erläuterten Gewährleistungsansprüche gelten nur
dann,wenn dasProdukt gemäß denvon AmershamBiosciences
geliefertenAnweisungeninstalliertund benutzt wurde.
Amersham Biosciencces übernimmt keinerlei Haftung fürSchädenoder
Folgeschäden, einschließlich, aber nicht begrenzt auf Gewinneinbußen, Einkom-
mensverluste,entgangeneGeschäftsabschlüsse,VerlustderGebrauchsfähigkeit
oder andere Verluste, die wie auch immer durch eine fehlerhafte oder
unsachgemäßeVerwendung desProduktsverursachtwurden.
©1998AmershamBiosciences Limited.
Alle Rechte vorbehalten.
Die vorliegende Veröffentlichung darf nur mit vorhergehender schriftlicher Gene-
hmigung durch das Unternehmen vervielfältigt, in einem Abrufsystem gespe-
ichertoderin irgendeinerForm odermit irgendwelchenMitteln übertragen
werden.
Informazioni importanti per l’operatore
Per un utilizzo sicuro del prodotto, leggere attentam-
ente l’intero contenuto del presente manuale.
Il punto esclamativo all’interno di un
triangolo equilatero indica all’operatore la presenza di importanti
istruzioni di funzionamento e manutenzione nella documentazione
allegata al prodotto.
Si prega di inviare eventualicommential presentemanuale a:
Amersham Biosciences Inc.
Marketing Department
654 Minnesota Street
San Francisco, CA 94107 USA
Amersham Biosciecnes si riserva il dirittodi apportaremodificheai dati
tecnici senza preavviso.
Garanziae responsabilitá
Amersham Biosciences garantiscecheprimadellaconsegna il prodottoè
stato collaudato a fondo per soddisfare i requisiti specificati. La garanzia inclusa
nelle condizioni di consegna risulta valida solamente se il prodotto è stato instal-
lato edutilizzatonel rispetto delleistruzioni fornitedaAmersham
Biosciences
Amersham Biosciences non potrà essereritenuta responsabiledi incidenti
o danni consequenziali, inclusi’ma non limitati’a perdite di profitti, mancato
guadagno, perditedi affari,difetti di funzionamento erelativeesposizioni, dovuti
ad un utilizzo non corretto del prodotto.
©1998AmershamBiosciences Limited.
Tutti i diritti riservati.
Nessuna parte della presente pubblicazione può essere riprodotta, conservata in
sistemi di gestione dati o trasmessa in alcun forma né per nessuno scopo senza
autorizzazione scritta del produttore.
Français
Español
Deutsch
Italiano

Hoefer DALT System
AmershamBiosciences
CONTENTS
Hoefer DALT System Function and Description 3
DALT System Components 3
Unpacking 7
Important Information/Informations Importantes 8
Specifications 9
Required or Convenient, But not Supplied 11
Preparing the Gel Caster 12
Casting Homogeneous (Non-Gradient) Gels 14
Preparation for Gradient Gel Casting 16
Configuring the Gradient Divider 16
Calibrating the Peristaltic Pump 17
Casting Gradient Gels 18
Gradient Casting Setup 18
Pouring Gel Solutions for Gradient Gels 19
Applying Overlay to DALT Slab Gels 22
Polymerization 22
Unloading the Gel Caster 23
Preparing the DALT Tank for Electrophoresis 24
Connecting a Refrigerated Circulating Bath 24
Filling the Tank with SDS Electrophoresis Buffer 24
Loading and Running Second Dimension Gels 27
Equilibrating IPG Strips 27
Loading the IPG Strips onto the DALT Slab Gels 29
Loading Cassettes into the DALT Tank 31
Electrophoresis Conditions in the DALT Tank 32
Unloading the DALT Cassettes 33
Cleaning DALT Cassettes 33
The DALT Blotting Kit 34
Preparing the DALT Tank for Transfer 34
Assemble the Transfer Cassette 35
Loading the Cassettes 37

Hoefer DALT System
2AmershamBiosciences
Connecting the Power Supply 38
Transfer Conditions 39
Completing the Transfer 39
Factors Affecting the Transfer 40
Recipes 41
Homogeneous Gel Solutions 46
Gradient Gel Solutions 49
For 25 gels, 1 mm 50
For 12 gels, 1 mm 51
For 23 gels, 1.5 mm 52
For 12 gels, 1.5 mm 53
Gel Identification Numbers 54
Care and Maintenance 55
Cleaning 55
Removing the Electrode Panels from the DALT Tank 55
Troubleshooting 56
References 60
Customer Service Information 61
Technical Service and Repair 61
Ordering information 61
Index 63

Hoefer DALT System Hoefer DALT System Function and Description
Amersham Biosciences 3
Hoefer DALT System Function and Description
In 2-D protein electrophoresis, proteins are separated first according to isoelectric point
by isoelectric focusing, most reliably on immobilized pH gradient (IPG) strips. The
second dimension electrophoresis separates the proteins on the basis of their molecular
mass on a slab gel containing the denaturing detergent, sodium dodecyl sulfate (SDS).
The Hoefer DALT System is designed to simplify the handling of multiple second
dimension gels and improve the reproducibility of the second dimension separation.
DALT System Components
The Hoefer DALT System comprises:
• a multiple vertical slab electrophoresis tank
• a multiple gel caster
• a gradient maker with peristaltic pump
• gel cassettes
• a blotting kit
Electrophoresis Tank
Figure 1. The DALT
Electrophoresis Tank
The DALT Electrophoresis Tank accommodates up to ten 23 ×19 cm slab gels for
separation under identical conditions. The sample, following focusing in an IPG strip, is
attached to the cathodic surface of the gel by embedding in agarose, then the gel is
turned 90° and run in a “sideways” orientation, with the IPG strip standing vertically at
one edge.
The tank is divided into three chambers by silicone rubber flap seals on the barrier
combs. The seals are not designed to be liquid-tight but they do provide a good barrier
to electrical current, causing most current to flow through the gels. Although there is
Hinged lid
Platinum-core
electrode panels (two)
Barrier combs (two) with
silicone rubber flap seals
Buffer
circulation flutes
Alumina
heat exchanger
High-voltage leads
(Siphon pump included,
but not shown)

Hoefer DALT System Function and Description Hoefer DALT System
4AmershamBiosciences
some leakage current bypassing the gels, this has little effect on system operation. The
barriers can be raised or removed by loosening the nylon screws which secure them in
place.
The left and right chambers contain platinum wire electrodes, cathode (–) and
anode (+). The center chamber provides cooling via circulating buffer and a tube-type
heat exchanger. Since the gel cassettes are almost entirely exposed to the buffer in the
center chamber, with only their ends protruding into the side chambers, cooling of the
center chamber buffer provides excellent temperature control of the gel slabs during the
run. Electrode panels can be removed by loosening the nylon screws securing them in
place. Removal is not typically necessary unless the platinum wire becomes broken and
requires replacement. See “Removing the Electrode Panels from the DALT Tank” on
page 55.
Figure 2. Rear view of
electrophoresis tank
Buffer moves into the pump through the middle of the three flutes running along the
length of the tank’s center chamber, and is expelled back into the tank through the left
and right flutes. The holes in the output flutes are aligned to generate maximum
circulation over both the cassettes and the heat exchanger on the floor of the tank's
centre chamber (beneath the flutes). As described on page 24, run the pump only when
the tank is filled with liquid, to prevent damage to the pump.
The Quick-fit connectors on the ends of the tank port tubing provide a leak-proof seal
when disengaged, eliminating spillage of coolant solution.
Pump shield, with foot
for open lid support
Buffer
circulation pump
Pump priming line*
*WARNING The liquid in the tank must cover the opening to the pump priming
line when the circulation pump is in operation.
Quick-fit connectors,
attach to circulating
water bath

Hoefer DALT System Hoefer DALT System Function and Description
AmershamBiosciences 5
Gel Cassettes
Figure 3. DALT gel
cassettes
The DALT gel cassettes are pre-assembled. Two glass plates are held together along one
edge by a strip of silicone rubber, and the glass spacers (1.5 or 1.0 mm thick) are glued
in position. To complete assembly, close the two plates like a book. Gels are removed by
opening the book after the run and lifting out the slab. The cassette is easily cleaned as a
unit, and can be stood upright in the open to dry. The cassettes are dishwasher-safe.
Cassettes are 20.4 ×25.5 cm and produce a gel about 19 ×23 cm: 46 ml for 1-mm
thick, 68 ml for 1.5-mm thick. The gels make the best use of the standard 20 ×25 cm X-
ray film.
Multiple Gel Caster
Figure 4. The DALT Gel
Caster
The DALT Gel Caster holds up to 25 gel cassettes, 1 mm thick, or up to 23 cassettes,
1.5 mm thick, with separating sheets, for casting homogeneous or gradient gels. Fewer
cassettes can be filled at one time by inserting filler blocks to occupy unneeded volume.

Hoefer DALT System Function and Description Hoefer DALT System
6AmershamBiosciences
A removable front plate and cassette separating sheets simplify loading and unloading
cassettes from the unit. A hydrostatic balance chamber allows accurate gradient
positioning and provides a means to flush the fill tubing before polymerization occurs.
The chamber also provides a “make up solution” to accommodate the volume
shrinkage during polymerization.
Pump-Assisted Gradient Maker
Figure 5. The DALT
Gradient Maker
The DALT Gradient Maker casts gradients of arbitrary shape, up to 2.0 liters total
volume. The solution is delivered at a controlled rate by a peristaltic pump. A flexible
liquid-tight divider partitions the gradient maker into two chambers of complementary
shapes for casting both linear and non-linear gradients. With the divider adjusting rod,
you can configure the gradient divider to adapt to your gradient gel requirements.
Divider adjusting rod

Hoefer DALT System Hoefer DALT System Function and Description
AmershamBiosciences 7
Blotting Kit
Figure 6. The DALT
Blotting Kit
The DALT Blotting Kit consists of a five-place acrylic cassette-holding rack and five
transfer cassettes, complete with cassette packing material— sponges and blotting paper.
The cassettes hold the gel and the transfer membrane secure, with just enough pressure
to assure even transfer.
Unpacking
Unwrap all packages carefully and compare contents with the packing list, making sure
all itemsarrived.Ifany partis missing,contactyourlocalAmersham Biosciences
sales office. Inspect all components for damage that may have occurred while the unit
wasin transit. Ifany partappears damaged,contactthe carrierimmediately. Be sureto
keep all packing material for damage claims or to use should it become necessary to
return the unit.
Rinse the DALT Tank and Gel Caster with distilled water and let air dry. Before initial
use, carefully clean glass plates and spacers with a dilute solution of laboratory cleanser
to remove fingerprints. Rinse thoroughly with tap and distilled water. See “Care and
Maintenance” on page 55 for more detailed instructions.

Hoefer DALT System
Amersham Biosciences
Important Information
• Plug the instruments into a properly grounded outlet.
• The safety lid must be in place before connecting the
power leads to a power supply.
• Turn all power supply controls off and disconnect the
power leads before opening the safety lid.
• Rinse only the electrodes (not the banana plugs) with
distilled water before and after use.
• Always disconnect the power cord before servicing.
• Do not operate the circulation pump if the DALT Tank is
empty. The pump is not self-priming and can be damaged
if run dry.
• Circulate only water or 50/50 water/ethylene glycol
through the heat exchanger. Never introduce anti-freeze
or any organic solvent into any part of the instrument.
Organic solvents will cause irreparable damage to the
unit!
• Do not connect the heat exchanger to a water tap or any
coolant source where the pressure is unregulated.
• Do not operate with buffer temperature above 45 °C. All
plastic parts are rated for 45 °C continuous duty.
Circulate coolant through the heat exchanger during
electrophoresis to minimize heating. Overheating will
cause irreparable damage to the unit!
For longer runs you can control heating somewhat by
chilling the buffer before use, running the unit in a cold
room, or both.
• Do not lift the DALT Tank by the buffer pump.
• Do not autoclave or boil this unit or any of its parts.
• The casting unit, when filled with glass plates and gel
solutions, is very heavy. Use caution when trying to move
or lift the casting unit.
• If this equipment is used in a manner not specified by the
manufacturer, the protection provided by the equipment
may be impaired.
• Only accessories and parts approved or supplied by
Amersham Biosciences may be usedfor operating,
maintaining, and servicing this product.
Informations Importantes
• Raccorder l’instrument à une prise de terre appropriée.
• Le couvercle de sécurité doit être en place avant de
brancher les prises au générateur.
• Eteindre le générateur et débrancher les prises avant
d’ouvrir le couvercle de sécurité.
• Rinser seulement les électrodes (pas les "banana-plugs")
avec de l'eau distillée avant et après l'utilisation.
• Toujours déconnecter le cordon d’alimentation avant de
réparer l’instrument.
• Ne pas faire fonctionner la pompe de circulation tampon,
lorsque la cuve d'électrophorèse DALT est vide. La pompe
ne s'amorce pas automatiquement et peut-être
endommagée si elle tourne à sec.
• Faire circuler seulement de l’eau ou 50/50 d’eau et
d’éthylène glycol dans l’échangeur vertical à cirulation
d’eau. Ne jamais utiliser d’anti-gel ou tout autre solvant
organique avec cet instrument. Les solvants organiques
causeraient des dommages irréparables à l’appareil.
• Ne pas connecter l’échangeur vertical à circulation d’eau à
un robinet ou quelque source de refroidissement dont la
pression n’est pas régulière.
• Ne pas utiliser avec un tampon à une température au
dessus de 45 °C. Toutes les piéces en plastique sont
prévues pour résister à une température constante de
45 °C.
Faire circuler l’eau dans l’échangeur vertical durant
l’électrophorèse pour minimiser l’échauffement afin
d’éviter des dommages irréparables à l’instrument.
Pour des coulages plus long, on peut aussi contrôler la
température en refroidissant le tampon avant l’utilisation
et/ou en utilisant l’instrument dans une chambre froide.
• Ne pas soulever la Cuve DALT en s'appuyant sur la
pompe.
• Ne pas autoclaver ou stériliser cette unité, ni aucune de ses
pièces détachées.
• L'unité de coulage des gels, quand elle comprend les
plaques de verre et la solution d'acrylamide, est très
lourde. Prenez toute vos précautions,quand vous déplacez
ou soulevez l'unité.
• Si l'instrument n'est pas utilisé en conformité avec les
recommandations du fabriquant, les protections de
sécurité qui équipent cet appareil peuvent être rendues
inéfficaces.
• Seulement les accessoires et piéces detachées approuvés ou
fournisparAmershamBiosciences sont
recommandés pour l’utilisation, l’entretien et réparation
de cet appareil.

Hoefer DALT System Hoefer DALT System Function and Description
AmershamBiosciences 9
Specifications
DALT Electrophoresis Tank with Buffer Circulation Pump
Gel capacity, 1.0 or 1.5 mm thick 10 gels
Electrophoresis buffer volume 20 liters
Blotting buffer volume 22 liters
Dimensions (h ×w ×d) lid closed: 38 ×48 ×33 cm (15 ×19 ×13 in)
lid open: 50 ×48 ×33 cm (20 ×19 ×13 in)
Weight 16.8 kg
Maximum wattage 200 W
Maximum voltage 500 V DC
Maximum amperage 1000 mA
Maximum temperature 30 °C
Environmental operating conditions Indoor use: 4 – 40 °C
Humidity up to 90%
Altitude up to 2000 m
Installation category II
Pollution degree 2
Buffer circulation pump rate 20 liters/min
115 V~ 50/60 Hz, 2.4 A, 180 W
230 V~ 50/60 Hz, 1/0.83 A
Product certifications* EN61010-1, UL3101-1, CSA C22.2 1010.1, CE
DALT Multiple Gel Caster
Gel Capacity
1.0 mm thick
1.5 mm thick
25 gels
23 gels
Acrylamide solution volume (total) 1,800 ml
Dimensions (h ×w ×d) 29 ×34 ϖ25 cm
Weight 33.5 kg
DALT Gel Cassettes
Glass cassette dimensions (w ×h) 25.5 ×20.4 cm
Slab gel dimensions 1.0 mm ×23.4 ×19.5 cm
1.5 mm ×23.4 ×19.5 cm

Hoefer DALT System Function and Description Hoefer DALT System
10AmershamBiosciences
DALT Gradient Maker
Dimensions (h ×w ×d) 54 ×19 ×18 cm
Maximum gradient volume 2,000 ml
Weight 5.4 kg
Peristaltic Pump for Gradient Maker
115 V~ 37 W, 1.5A
230 V~ 37 W, 0.9A
Weight 4.1 kg (9 lbs.)
Dimensions (h ×w ×d) 13.5 ×18 ×22 cm
Environmental operating conditions Indoor use: 0 – 40 °C
Humidity: 10 – 90%
Altitude: up to 2000 m
Installation category II
Pollution degree 2
Product certifications* EN61010-1, UL508, cUL (115 V), IEC 1010 (230 V),
CE
DALT Blotting Kit
Capacity 5 gels
Rack dimensions (h ×w ×d) 28 ×23 ×26 cm
Weight 3.2 kg
*This declaration of conformity is only valid for the instrument when it is:
• used in laboratory locations,
•used as deliveredfrom AmershamBiosciences,exceptfor alterations
described in the User Manual, and
• connected to other CE-labeled instruments of products recommended or
approvedby AmershamBiosciences.

Hoefer DALT System Hoefer DALT System Function and Description
AmershamBiosciences 11
Required or Convenient, But not Supplied
Refrigerated Cooling Bath
DALT Tank cooling is provided by circulating chilled liquid through the heat exchanger
on the floor of the central chamber. With the aid of Quick-fit connectors, you can
connect a refrigerated circulating bath, such as the MultiTemp III, to the fittings at the
back of the DALT Tank. We strongly recommend active cooling for protein transfers.
Focused IPG Strips
Immobiline DrySrips provide a stable gradient pH gradient (IPG) immobilized onto an
acrylamide matrix and supported by a plastic film backing. They are readily available,
easy to handle and not prone to breakage and stretching. IPG strip focusing is
accomplished on the IPGphor or Multiphor II with the Immobiline DryStrip tray
accessory.
Power Supply
For overnight or full-day runs, a conventional electrophoresis power supply delivering
600 V at 400 mA, such as the EPS 601, is suitable. For multiple tanks or faster runs (8 –
9 h) a higher current power supply, such as the EPS 2A200, is desirable.
Transfer Membranes
The DALT Blotting kit includes 50 pieces of blotting paper. For electrophoresis tank
blotting, Amersham Biosciences offersa completeline oftransfer membranes,
includingpure nitrocellulose, supported nitrocellulose, PVDFand nylon. Choose the
appropriate transfermembrane foryourapplication.
Small Gadgets
•A small plastic spatula or thin plastic ruler to insert and seat IPG gels on the tops of
the slab gels between the glass plates
•Plastic-covered dish racks (Rubbermaid or equivalent) for holding the DALT gel
cassettes during the loading process
•A microwave oven or 100 °C heating block for preparing the agarose sealing
solution
•A Wonder Wedge (80-6127-88) for prying open the gel cassettes after the DALT run
• Screw-cap culture tubes, 25 ×200 mm, for storing IPG strips at –40 °C
• GelSeal for lubricating the gradient gasket, to assure a leakproof seal
•A peristaltic pump is recommended for more rapid removal of buffer than the small
siphon pump supplied with the DALT Tank. The pump can be set up to deliver used
buffer that is not radioactive directly to a lab drain.
•A vacuum pump for degassing solutions
• Thick, latex dishwashing gloves, with textured fingers

Preparing the Gel Caster Hoefer DALT System
12AmershamBiosciences
Preparing the Gel Caster
Set up the gel caster near a sink, in a tray or container that can act as a catch basin for
any liquid that may overflow the unit or empty out of it when you open it to remove the
gels. Alternatively, place the unit on a plastic or rubber kitchen drain board that empties
into a sink.
The DALT Gel Caster can accommodate up to twenty-five 1-mm, or twenty-three
1.5-mm gel cassettes, with separator sheets between each cassette, before the first
cassette and after the last cassette. If you are not preparing the maximum number of
gels, use filler blocks and separator sheets to take up the excess caster volume. Place
separator sheets between the filler blocks.
Cast at least 22 cassettes for two 10-sample runs to allow for the possibility of one or
two imperfect gels.
1. Check that the caster is level. Remove the front plate and rest the caster on its back,
with its feet facing you. Place the triangular sponge in the base of the V-shaped feed
channel.
Plates are more easily loaded in this orientation. Check that the caster is clean, and
free of dust.
2. Lubricate the foam sealing gasket with a light coating of GelSeal to help assure leak-
proof sealing. Place the gasket in the groove along the front side of the caster. Avoid
stretching the gasket.
3. Start filling the gel caster by placing a separator sheet against the inside wall to
make it easier to remove the cassettes and filler blocks after polymerization.
If you are casting fewer than the maximum number of gels, first add a selection of
filler blocks, with a separator sheet between each block. For example, to cast twelve
1.0-mm gels, you may need four 25-mm blocks and one 3-mm block, with
separator sheets between each block. To cast twelve 1.5-mm gels, you may need
three 25-mm blocks, one 12-mm block, one 6-mm block, and one 3-mm block,
with separator sheets between each block.
Next add the cassettes, with all hinge strips vertical and aligned on the side of the
unit opposite the feed tube. Insert a gel separator sheet between each cassette pair.
End with a separator sheet between the final cassette and the gel caster face plate. If
necessary, use additional separator sheets to bring the level of the stack even with
the edge of the caster.
4. Put the removable front plate of the gel caster in place and screw on the black
knurled knobs. Tighten these hand tight (not over-tight), then carefully place the gel
caster in an upright position.
Be sure the sealing gasket on the front plate forms a tight seal against the face plate.

Hoefer DALT System Preparing the Gel Caster
AmershamBiosciences 13
5.Prepare asetofgel labelsonfilter paper, asdescribed on page53.
Cut the labels apart, leaving little excess around the characters, and place them in
order in front of the gel caster. Then, taking care to keep track of which cassette will
be numbered next, drop the numbers, in order, into the cassettes, on the side
opposite the gradient inlet port. As the gradient is introduced, the numbers fall to
the floor of the caster but remain in the respective cassettes and ultimately are
polymerized into the gels.
6. Insert the end of the plastic feed tube, supplied with the gel caster, into the grommet
in the floor of the side hydrostatic balance chamber of the caster (Figure 7).
Figure 7. Attaching the
feed tube to the gel caster
The feed tube must be snugly in place so that there is no leakage from the balance
chamber into the caster at this point. The feed tube should be connected directly to
the gradient maker tubing or to a funnel with flexible vinyl tubing.
Grommet
Feed tube
V-well
Balance chamber
Identification number

Casting Homogeneous (Non-Gradient) Gels Hoefer DALT System
14AmershamBiosciences
Casting Homogeneous (Non-Gradient) Gels
WARNING Acrylamide is a neurotoxin. Always use mechanical pipettes and wear
protective gloves when working with acrylamide solutions, IPG strips
or surfaces that come in contact with acrylamide solutions.
1. Be sure the entire gel casting system is clean, dry, and free from any polymerized
acrylamide.
2. Prepare a sufficient volume of gel overlay solution (water-saturated n-butanol). You
need 0.75 ml of overlay for each cassette, or about 20 ml for a set of 25 cassettes.
3. Make up 200 ml of displacing solution (page 42).
4. Make up the gel acrylamide stock solution, without adding the 10% ammonium
persulfate (APS) or 10% N,N,N',N',-tetramethylethylenediamine (TEMED). See
“Homogeneous Gel Solutions” on page 46.
5. Load the gel caster with cassettes, separator sheets and filler blocks, if necessary.
Place a gel label in each cassette. See page 12 for directions.
6. Connect the feed tube to a funnel held in a ring-stand at a level about 12 inches
above the top of the gel caster (Figure 8). Insert the other end of the feed tube in the
grommet in the bottom of the balance chamber.
Figure 8. Setting up the
caster for a non-gradient
gel
7. Load the balance chamber with 150 ml heavy displacing solution.
8. Add the appropriate volumes of APS and TEMED only when you are ready to pour
the gel, not before.
Once these reagents are added, polymerization begins. You have about 10 minutes
before the gels begin to solidify. Vary the amount of TEMED added to control the
rate of polymerization.
9. Slowly pour the gel solution into the funnel, taking care to avoid introducing any
bubbles into the feed tube line.
Funnel
Grommet
Feed tube
Balance chamber

Hoefer DALT System Casting Homogeneous (Non-Gradient) Gels
AmershamBiosciences 15
10. Once the pouring is complete, remove the feed tube from the balance chamber
grommet.
As soon as the feed tube is removed, the dense blue displacing solution flows down
the connecting tube to the unit, fills the V-well and the sloped trough at the bottom
of the caster. If the V-well is not completely filled and the level of gel in the casettes
is more than 1 cm below the top of the cassettes, you may add up to 50 ml more
displacing solution to the balance chamber.
The displacing solution pushes the remaining acrylamide solution out of the V-well
into the gel cassettes. As gels contract during polymerization, the displacement
solution is drawn in from the bottom.
IMPORTANT Apply overlay immediately! See “Applying Overlay to DALT Slab
Gels” on page 22.
11. Allow non-gradient gels to polymerize for at least 1 hour.

Preparation for Gradient Gel Casting Hoefer DALT System
16AmershamBiosciences
Preparation for Gradient Gel Casting
Successful gel casting requires planning, timing and practice.
A full DALT Gel Caster requires approximately two liters of acrylamide stock.
Polymerization begins as soon as you add TEMED and APS to the acrylamide stock.
Once you have added these reagents, there is no time to adjust the gradient maker
divider, or the cassettes and separators in the gel caster.
To familiarize yourself with the gel caster and gradient maker before casting gels, we
recommend you set up the unit, as described on page 12, and configure the gradient
divider, as described on this page. Follow the gradient pouring procedure on page 18,
substituting water for the appropriate volume of light solution, and a mixture of
glycerol and water for the appropriate volume of heavy solution.
When the angle of the gradient divider is correctly adjusted and you are comfortable
with the gel casting procedure, clean all parts of the caster and gradient maker, including
sponges, separating sheets and filler blocks, with a solution of mild detergent, followed
by a fresh water rinse.
Configuring the Gradient Divider
You can adjust the shape of the gradient divider to suit the number of gels to be cast and
shape of the gradient you want. When casting a full tank of gels, the angle of the
adjustable divider to the floor of the gradient maker should be about 70 °. For fewer
gels, decrease the divider angle (Figure 9). Whatever angle you use, a straight divider
gives a linear gradient.
Figure 9. Gradient divider
configurations for half-full
and full Gel Caster tanks.
1. Determine the amount of gel solution needed to cast the desired number of gels.
2. Loosen the faceplate screws on the empty gradient maker to adjust the gasket angle.
Refer to the divider configurations in Figure 9 as a guideline to the angle you should
use.
Pull the divider slightly to help move it into position. Use the red adjuster rod
provided with the gradient maker to push the gradient divider down.
Divider angle (approx.) 40 ° 50 ° 60 ° 70 °
Number/thickness 12 gels/1.0 cm 12 gels/1.5 cm 25 gels/1.0 cm 23 gels/1.5 cm