GE HEALTHCARE Ettan DALTsix User manual

Ettan DALTsix
Electrophoresis System
User Manual
GE Healthcare
80649249AC.book Page 1 Monday, November 13, 2006 2:42 PM

80649249AC.book Page 2 Monday, November 13, 2006 2:42 PM

Important user information
All users must read this entire manual to fully understand the safe use of
Ettan DALTsix Electrophoresis System.
WARNING!
The WARNING! sign highlights instructions that must be
followed to avoid personal injury. It is important not to
proceed until all stated conditions are met and clearly
understood.
CAUTION!
The CAUTION! sign highlights instructions that must be followed to avoid
damage to the product or other equipment. It is important not to proceed
until all stated conditions are met and clearly understood.
Note
The Note sign is used to indicate information important for trouble-free
and optimal use of the product.
CE Certifying
This product meets the requirements of applicable CE-directives. A copy of
the corresponding Declaration of Conformity is available on request.
The CE symbol and corresponding declaration of conformity, is valid for
the instrument when it is:
– used as a stand-alone unit, or
– connected to other CE-marked GE Healthcare instruments,
or
– connected to other products recommended or described in this manual,
and
– used in the same state as it was delivered from GE Healthcare except for
alterations described in this manual.
Recycling
This symbol indicates that the waste of electrical and
electronic equipment must not be disposed as unsorted
municipal waste and must be collected separately. Please
contact an authorized representative of the manufacturer for
information concerning the decommissioning of your
equipment.
WARNING!
This is a Class A product. In a domestic environment this product may
cause radio interference in which case the user may be required to take
adequate measures.
• Connect the instrument to a properly grounded electrical outlet.
• The safety lid must be firmly in place before power can be applied.
• Turn off the power to the gels before opening the safety lid.
• Do not connect the heat exchanger to a water tap or any other water
source with unregulated pressure that may exceed 82 kPa (12 PSI).
• Rinse and flush the tank and pumping system with distilled or
deionized water before and after use.
• Always disconnect the power cord before servicing.
• Do not run the circulation pump if the electrophoresis tank is empty.
• Do not operate with buffer temperature above 40 ºC. All plastic parts
are rated for 40 ºC continuous duty.
• Turn the buffer circulation pump on during electrophoresis to
minimize uneven heating even if not connected to a thermostatted
circulator.
• Connect the heat exchanger to an external thermostatted circulating
bath. Overheating will cause irreparable damage to the unit.
• Do not autoclave or boil this unit or any of its parts.
• Use care when lifting and moving the electrophoresis unit. It is best to
move the unit when empty.
• The casting unit, when filled with glass plates and gel solutions, is very
heavy. Use caution when trying to move or lift the caster.
• The protection provided by the equipment may be impaired if this
equipment is used in a manner not specified by the manufacturer.
• Only accessories and parts approved or supplied by GE Healthcare
may be used for operating, maintaining and servicing this product.
Unpacking, inventory, and set-up
Unwrap all packages carefully and compare contents with the packing list,
making sure all items arrived. If any part is missing, contact your local sales
office. Inspect all components for damage that may have occurred while
the unit was in transit. If any part appears damaged, contact the carrier
immediately. Be sure to keep all packing material for damage claims or to
use should it become necessary to return the unit.
Heat exchanger
The heat exchanger is built into the base of the separation tank. The white
alumina ceramic heat exchange surface is fragile and the user should
avoid dropping anything directly on surface.
When connected to a circulator bath, coolant passes through a serpentine
chamber beneath the ceramic plate. The thin ceramic permits rapid heat
exchange between the coolant and the buffer in the electrophoresis tank.
The ceramic plate is attached with silicone rubber adhesive. The heat
exchanger tubing connectors are 13-mm o.d.
The heat exchanger is rated to a maximum of 0.8 atmospheres above
ambient (12 psig). Do not connect it to a water tap. Connect only to coolant
sources with regulated pressure.
To connect the coolant tubing lines to the tank, remove the lid and the anode rack from
the tank and lay the tank fill-label (front) side down. Guide the free ends of the coolant
tubes through the holes in the tank base and slip them onto the plastic connector
fittings, then pull the collector collar onto the tubing to lock it in place. The end of the
tubing can be lubricated with glycerol or a mix of glycerol:water to ease the fit.
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Wichtige informationen
• Schliessen Sie das Gerät ausschliesslich an eine geerdete Steckdose
an.
• Der Sicherheitsdeckel muss korrekt sitzen bevor die elektrische Span-
nung angelegt werden kann.
• Schalten Sie die elektrische Verbindung zu den Gelen ab bevor Sie den
Sicherheitsdeckel öffnen.
• Schliessen Sie den Wärmetauscher niemals an eine Wasserleitung
oder eine unkontrollierbare Druckquelle an, welche 82 kPa (12 PSI)
übersteigen kann.
• Vor und nach jedemGebrauch: Waschen und spülen Sie den Puffertank
und das Pumpensystem mit destilliertem oder entionisiertem Wasser.
• Vor Reinigung oder Reparatur der Kammer: Entfernen Sie stets das
Stromkabel vom Netz.
• Betreiben Sie die Pumpe nie bei leerem Puffertank.
• Verwenden Sie das Gerät nie mit einer Puffertemperatur über 40 ºC.
Alle Plastikteile sind für Temperaturen unter 40 ºC spezifiziert.
• Lassen Sie während der Elektrophorese die Pufferumwälzpumpe auch
dann laufen, wenn die Kammer nicht an einen Kühlthermostaten an-
geschlossen ist: Dies minimiert eine ungleichmässige Temperatur-
verteilung.
• Verbinden Sie den Wärmetauscher mit einem externen Kühlthermo-
staten. Überhitzung verursacht eine irreparable Schädigung des
Gerätes.
• Autoklavieren Sie niemals das Gerät oder Teile davon.
• Üben Sie Vorsicht wenn Sie das Gerät anheben und transportieren. Am
besten transportieren Sie das Gerät nur im leeren Zustand.
• Die Gelgiesseinheit ist sehr schwer, wenn sie mit Glasplatten und Gellö-
sung gefüllt ist. Üben Sie Vorsicht, wenn Sie versuchen die Giesseinheit
anzuheben oder zu transportieren.
• Der Sicherheitsschutz des Gerätes kann unter Umständen nicht funk-
tionieren, wenn das Gerät in einer Weise benützt wird, für die es vom
Hersteller nicht spezifiziert wurde.
• Nur Originalzusatzteile und Ersatzteile, die vom GE Healthcare geneh-
migt oder geliefert wurden, dürfen zum Betrieb, zur Erhaltung und zur
Reparatur dieses Produktes verwendet werden.
Renseignements importants d'utilisation
• Brancher l'appareil à une prise de courant électrique correctement
reliée à la terre.
• Le couvercle de sécurité doit être parfaitement en place avant de
mettre l'appareil sous tension.
• Eteindre le générateur et débrancher les prises avant d'enlever le cou-
vercle de sécurité
• Ne pas connecter l'échangeur de chaleur (vertical) à circulation d'eau
à un robinet ou quelque source d'eau dont la pression est non regulée
et pourrait dépasser 82 kPa (12PSI).
• Rincer et vider la cuve d'électrophorèse et le système de pompage
avec de l'eau distillée ou déionisée avant et après utilisation.
• Débrancher toujours la prise avant tout entretien.
• Ne pas allumer la pompe de circulation lorsque la cuve d'électro-
phorèse est vide
• Ne pas utiliser avec un tampon à une température au dessus de 40 °C.
Toutes les pièces en plastique sont prévues pour résister à une
température constante de 40 °C.
• Allumer la pompe de circulation du tampon pendant l'électrophorèse
afin de réduire au minimum un échauffement non uniforme, même si
non reliée à un bain à circulation d'eau et à thermostat.
• Relier l'échangeur de chaleur (vertical) à circulation d'eau à un bain à
circulation d'eau et à thermostat. La surchauffe endommagera l'unité
de façon irréparable.
• Ne pas autoclaver ou faire bouillir cet appareil, ni aucunes de ses pièc-
es détachées.
• Soulever et déplaçer l'unité d'électrophorèse avec beaucoup de soin. Il
vaut mieux déplacer l'unité une fois vide. L'unité de coulage des gels,
une fois remplie de plaques de verre et de solutions de gel, est très
lourde. Soulever et déplacer l'unité de coulage des gels avec beaucoup
d'attention.
• La protection fournie par cet équipement peut etre altérée si l'équipe-
ment est utilisé d'une façon non indiquée par le constructeur.
• Seuls les accessoires et piéces detachées approuvés ou fournis par
GE Healthcare sont recommandés pour l'utilisation, l'entretien et rép-
aration de cet appareil.
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Contents
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC 5
Contents
1 Ettan DALTsix Electrophoresis
System function and description
1.1 Ettan DALTsix Electrophoresis System function and description ............................................................................7
1.1.1 Electrophoresis Unit ........................................................................................................................................................7
1.1.2 Gel Caster .............................................................................................................................................................................7
1.1.3 Gel Casting Cassettes .....................................................................................................................................................8
1.1.4 Gradient Maker ..................................................................................................................................................................8
2 Specifications
2.1 Ettan DALTsix Electrophoresis Unit ........................................................................................................................................9
3 Preparing the Ettan DALTsix Gel Caster ....................................................................................11
4 Casting homogeneous gels .........................................................................................................................................13
5 Casting gradient gels
5.1 Preparation for gradient gel casting ..................................................................................................................................15
5.2 Gradient casting setup .............................................................................................................................................................. 15
5.3 Pouring gel solutions for gradient gels ..............................................................................................................................16
6 Unloading the gel caster ................................................................................................................................................ 19
7 Using Ettan DALTsix for electrophoresis
7.1 Preparing second dimension gels: equilibration and loading ................................................................................ 21
7.2 Preparing the electrophoresis unit ...................................................................................................................................... 22
7.2.1 Unloading the gels and cleaning the unit ..........................................................................................................24
7.3 Recommended running conditions ..................................................................................................................................... 24
8 Recipes ..................................................................................................................................................................................................25
9 Troubleshooting ........................................................................................................................................................................ 29
10 Care and maintenance
10.1 Cleaning ........................................................................................................................................................................................... 33
10.2 Replacement of components ................................................................................................................................................ 33
10.3 Recycling .........................................................................................................................................................................................33
11 Customer service information
11.1 Technical service and repair ..................................................................................................................................................35
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Contents
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Ettan DALTsix Electrophoresis System function and description 1
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1 Ettan DALTsix Electrophoresis
System function and description
1.1 Ettan DALTsix Electrophoresis System function and description
In 2-D electrophoresis, proteins are separated according to isoelectric point by isoelectric focusing, most reliably
on Immobiline™ DryStrip immobilized pH gradient (IPG) gel strips using the IPGphor™ or Multiphor™ II IEF
Systems. The second-dimension electrophoresis separates the proteins on the basis of their molecular mass
using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Ettan™ DALTsix
Electrophoresis System is designed to handle large second-dimension gels in a simple, efficient, and
reproducible manner.
1.1.1 Electrophoresis Unit
The Ettan DALTsix Electrophoresis Unit accommodates up to six 25.5 × 20.5 cm slab gels, either 1 mm or 1.5 mm
thick, in a common tank under identical conditions. A sample, focused in an IPG strip, is placed on the cathodic
(upper) surface of a slab gel and sealed in place with agarose. Up to six gel cassettes are inserted into the
electrophoresis unit and any unused slots are filled with blank cassette inserts. The Upper Buffer Chamber/Buffer
Seal (UBC) is pushed down over the cassettes, holding the gel cassettes in slots flanked by a double rubber
gasket.
Power is supplied to the unit by an external source such as an EPS 601 Power Supply. The unit is capable of
handling 600 V, 400 mA, or 100 W.
A pump mounted under the lower buffer chamber circulates the buffer, pumping it up into the chamber on the
right-hand side, between the cassettes, down the left side, and over the internal heat exchanger before returning
to the pump. The pump starts when it is plugged in. For temperature control, the heat exchanger located in the
bottom of the unit must be connected to a MultiTemp™ III or similar circulating water bath.
1.1.2 Gel Caster
The Ettan DALTsix Gel Caster holds up to six 1-mm or 1.5-mm gel cassettes, with separator sheets, for casting
homogenous or gradient gels. When desired, fewer gels can be cast at one time by using blank cassette inserts
to occupy any unneeded volume. The removable faceplate and separator sheets simplify loading and unloading
the casting unit. The groove in the back of the caster provides the channel through which the gel solution is
poured into the caster unless gradients are being the cast
by displacement through the lower tubing fitting.
Ettan DALTsix System components
• Six slot vertical slab electrophoresis unit
• Gel caster
• Gel casting cassettes
•Gradientmaker
Fig 1-1. Exploded view of Ettan DALTsix Electrophoresis Unit. Fig 1-2. Ettan DALTsix Gel Caster for casting 1.0 and 1.5 mm large format gels.
Lid with leads
Safety interlock
Upper buffer chamber
Anode assembly/
cassette carrier
Lower buffer chamber
Ceramic heat exchager
Buffer circulation pump
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1 Ettan DALTsix Electrophoresis System function and description
8Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
1.1.3 Gel Casting Cassettes
The Ettan DALTsix System uses the same cassettes as the Ettan DALTtwelve Electrophoresis System (see Fig 1-3).
The gel casting cassettes are preassembled. Two glass plates are joined along one edge by a hinge strip of
silicone rubber, and the vinyl side spacers (1.0 mm or 1.5 mm thick) are glued in place. To complete assembly,
close the two plates like a book and press the plates together over the length of the spacer. Gels are removed by
opening the book after the run and carefully lifting out the gel slab. Care must be taken to ensure that the gel
does not adhere to the spacers and tear during removal. The cassette is cleaned as a unit and can be stood
upright to dry. The cassettes can be cleaned in an automatic dishwasher. Cassettes are 27 × 22 cm and produce
a gel about 25.5 × 20.5 cm. A 1.0-mm thick gel has a volume of approximately 52 ml and a 1.5-mm thick gel has
a volume of approximately 78 ml.
Fig 1-3. Gel Casting Cassettes.
1.1.4 Gradient Maker
The Ettan DALTsix Gradient Maker is designed for producing linear gradients of aqueous solutions ranging in
volume from 200–2 000 ml. The gradient maker can be used to form convex and concave exponential gradients
with the addition of a one-holed rubber stopper, a piece of rigid tubing, and a piece of flexible tubing.
Fig 1-4. Ettan DALTsix Gradient Maker.
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Specifications 2
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC 9
2 Specifications
2.1 Ettan DALTsix Electrophoresis Unit
Maximum voltage 600 V DC
Maximum current 400 mA
Maximum power 100 W
Gel capacity 6 gels
Electrophoresis buffer volume 5.5 l
Dimensions (h × w × d) 40.3 × 54.2 × 16.0 cm
Weight (empty) 11.1 kg
Maximum temperature 40 ºC
Environmental operating conditions Indoor use, 4–40 ºC
Humidity up to 90%
Altitude to 2000 m
Installation category II
Pollution degree 2
115 V~ model 104–127 VAC, 60 Hz, 8 W
230 V~ model 207–264 VAC, 50 Hz, 7 W
Compliance with standards The declaration of conformity is valid for the instrument
only if it is:
• used in laboratory locations
• used in the same state as it was delivered from GE
Healthcare except for alterations described in the User
Manual
• connected to other CE labelled GE Healthcare modules
or other products as recommended.
Safety standards This product meets the requirement of the Low Voltage
Directive (LVD) 73/23/EEC through the following harmonized
standards:
• EN 61010-1
• IEC 61010-1
• CAN/CSA-C22.2 No. 61010-1
• UL61010-1
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2 Specifications
2.1 Ettan DALTsix Electrophoresis Unit
10 Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
EMC standards This device meets the requirements of the EMC Directive 89
336/EEC through the following harmonized standards:
• EN 61326 (emission and immunity)
• EN 55011, GR 1, Class A (emission)
• This device complies with part 15 of the FCC rules
(emission). Operation is subject to the following two
conditions:
1 This device may not cause harmful interference.
2 This device must accept any interference received,
including interference that may cause undesired
operation.
Ettan DALTsix Gel Caster
Gel capacity 6 gels, 1.0 mm or 1.5 mm thick
Acrylamide solution volume (total) 425 ml for 1.0 mm thick gels,
550 ml for 1.5 mm thick gels
Dimensions (h × w × d) 27 × 37 × 8 cm
Weight (empty) 3.6 kg
1.0 mm Gel Casting Cassette
Cassette dimensions (w × h × d) 27.6 × 21.7 × 0.70 cm
Slab gel dimensions (w × h × d) 25.5 × 20.5 × 0.10 cm
1.5 mm Gel Casting Cassette
Cassette dimensions (w × h × d) 27.6 × 21.7 × 0.75 cm
Slab gel dimensions (w × h × d) 25.5 × 20.5 × 0.15 cm
Ettan DALTsix Gradient Maker
Volume 1000 ml
Weight 0.73 kg
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Preparing the Ettan DALTsix Gel Caster 3
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3 Preparing the Ettan DALTsix Gel Caster
Set up the gel caster near a sink, in a tray, or on a drainboard so that any liquid that overflows, spills, or drains
out of the unit during pouring or disassembly can be easily contained.
The Ettan DALTsix Gel Caster accommodates up to six 1-mm or 1.5-mm gel cassettes with separator sheets
(0.5 mm) between them. If you are not planning to cast a full set of gels, use the blank cassette inserts (purchased
separately) with separator sheets between them to occupy the extra space.
Gel labels, for easy indexing of gels and samples, can be placed in the cassettes at any time during the assembly
of the caster.
1 Check that the caster is level. Remove the faceplate and lay the caster on its back. If casting gels by the
displacement method, remove the triangular wedge in the V-shaped base.
2 Fill the gel caster starting with a separator sheet against the back wall. The separator sheets make it easier
to remove the cassettes from the unit after polymerization. Fill the caster by alternating cassettes with
separator sheets. The rubber hinge should be on the left side of the caster, with the matching ends of the
cassette down. End with a separator sheet then use the thicker filler sheets (1.0 mm) to bring the level of the
stack of cassettes and spacers even with the edge of the caster.
3 Remove the gray foam seal from the groove in the faceplate and lubricate it with a light coating of GelSeal
compound to help ensure a liquid-tight seal. Place the gasket back in the groove on the faceplate. Avoid
stretching the gasket by seating it from the ends first, working toward the middle.
4 Turn two black knobbed screws into the two threaded holes across the bottom until they are well engaged
(two to three full turns). Carefully place the faceplate onto the caster with the bottom slots resting on their
respective screws. Clamp both sides of the faceplate with six spring clips and tighten the screws. Be sure the
sealing gasket is compressed evenly by the faceplate and forms a tight seal with the caster. Do not
overtighten the screws.
5 If casting using the filling channel in the back plate of the caster, be sure to plug the barbed fitting in the
faceplate. If casting using the displacement method, attach the tubing from the peristaltic pump to the
barbed fitting.
Fig 3-1. Gel caster with parts labelled.
filling channel
separating sheets
filler sheets
faceplate and
sealing gasket
gel cassette
cassette hinge
spring clamp (6)
clamp screw
filler port
and cap
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3 Preparing the Ettan DALTsix Gel Caster
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Casting homogeneous gels 4
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4 Casting homogeneous gels
WARNING! When using hazardous chemicals, take all suitable protective measures, such as wearing
protective glasses and gloves resistant to the chemicals used. Follow local regulations and instructions for
safe operation and maintenance of the system.
WARNING! Acrylamide is a neurotoxin. Never pipette by mouth and always wear protective gloves when
working with acrylamide solutions, IPG strips, or surfaces that come into 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 1.0 ml of overlay
for each 1-mm cassette and 1.5 ml for overlay for each 1.5-mm cassette. Mixing 1-mm and 1.5-mm
cassettes, in one casting is not advised.
3 If casting using the displacement method, make up 200 ml of displacing solution (0.375 M Tris-Cl pH 8.8, 30%
(v/v) glycerol, bromophenol blue).
4 For a full 6-gel, 1.5-mm cassette set, make up 600 ml of acrylamide gel stock solution without ammonium
persulfate (APS) or N,N,N',N'-tetramethylethylenediamine (TEMED). For a full 6-gel, 1-mm cassette set, make
up 450 ml of acrylamide gel stock solution as above. This amount of gel solution will provide you with
sufficient volume to cast gels using either the filling channel or a peristaltic pump.
5 Assemble the gel caster as described in the proceeding section; the caster should be placed on a level bench
or on a leveling table so that gel tops are level.
6 Add the appropriate volumes of APS and TEMED only when ready to pour the gels, not before. Once these
two components are added, polymerization begins and the gel solution should be completely poured within
10 min.
7 Pour the gel solution into the filling channel. If displacement casting is being used, the flow rate on the
peristaltic pump should be increased slowly to the desired speed to avoid introducing any air bubbles.
8 For displacement casting, pump the gel solution into the caster until it is about 7–11 cm below the final
desired gel height. Stop the flow of acrylamide and transfer the feed tube to the container holding the
displacement solution. Restart the pump to displace the gel solution in the tubing and the V-trough at the
bottom of the caster. The remaining acrylamide solution is forced into the cassettes to the final gel height.
When the final desired gel height is reached, stop the pump and clamp off the tubing near the barbed fitting
at the bottom of the caster.
For filling channel casting, pour the gel solution into the channel until the solution reaches the final desired
height, about 1 cm below the top edge of the short plate.
The amount of gel solution required in either case will be ~550 ml for six 1.5-mm gels and ~425 ml for six
1-mm gels.
9 Immediately pipette the water-saturated n-butanol onto each gel. Avoid wetting adjacent plastic surfaces
of the caster with n-butanol.
10 Allow the homogeneous gels to polymerize for at least one hour before disassembling the caster. If the
peristaltic pump outlet tubing was connected directly to the caster, it should not be removed until
polymerization is complete. Do not leave the water-saturated n-butanol on the gels overnight as long term
exposure of acrylic to butanol will damage the caster.
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4 Casting homogeneous gels
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Casting gradient gels 5
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC 15
5 Casting gradient gels
5.1 Preparation for gradient gel casting
Successful gradient gel casting requires planning, timing, and practice. A full cast with the Ettan DALTsix Gel
Caster requires 420–550 ml of acrylamide stock. Polymerization begins as soon as TEMED and APS are added to
the acrylamide stock. At this point there is no time to adjust the gradient maker, or the cassettes and separators
in the gel caster. To familiarize yourself with the gel caster and gradient maker before casting gels, you should
set up the unit and carry out a practice gradient cast, substituting water for the appropriate volume of light
solution, and a mixture of glycerol and water for the appropriate volume of heavy solution.
A pore gradient gel results from mixing varying proportions of two solutions of different acrylamide
concentrations and densities, a light solution and a heavy solution. The heavy gel solution contains glycerol and
a higher concentration of acrylamide. During the gradient pouring procedure, the mixing ratio of high
concentration acrylamide solution to low concentration solution gradually changes, with the heavier (high
concentration) solution sinking below the light solution. This generates a downward gradient of increasing
acrylamide (gel) percentage.
5.2 Gradient casting setup
WARNING! When using hazardous chemicals, take all suitable protective measures, such as wearing
protective glasses and gloves resistant to the chemicals used. Follow local regulations and instructions for
safe operation and maintenance of the system.
WARNING! Acrylamide is a neurotoxin. Never pipette by mouth and always wear protective gloves when
working with acrylamide solutions, IPG strips, or surfaces that come into contact with acrylamide solutions.
1 Be sure the entire gel casting system is clean, dry, and free from any polymerized acrylamide. Remove the
rubber insert from the base of the V-shaped feed channel of the caster. The caster should be placed on a
level bench to ensure that the gels and gradients are even and level.
2 Add a magnetic stir bar of the appropriate dimensions (20-30 mm long) to the mixing chamber and place
the unit on a magnetic stirrer. If volumes will be less than half the capacity of the unit, an identical stir bar
should be placed in the reservoir chamber as well to balance the displacement and prevent backflow into
the reservoir when the chambers are first connected.
3 Connect tubing to the outlet connector and pump, and adjust pump speed, if used. Connect the tubing to
the gel casting unit.
4 Close the connecting valve between the reservoir (heavy) and mixing (light) chambers and clamp off the exit
line.
5 Prepare a sufficient volume of gel overlay solution (water-saturated n-butanol). You need 1.0 ml of overlay
for each 1-mm cassette and 1.5 ml for each 1.5-mm cassette.
6 Make up 200 ml of displacing solution.
7 Make up the gel acrylamide solutions from the stock mixes, but do not add the 10% ammonium persulfate
(APS) and 10% N,N,N',N',-tetramethylethylenediamine (TEMED). See "Gradient gel solutions" on page 16.
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5 Casting gradient gels
5.3 Pouring gel solutions for gradient gels
16 Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
Fig 5-1. DALTsix gradient maker.
5.3 Pouring gel solutions for gradient gels
1 Prepare the gel caster, as described on page 6, and place identifying gel labels inside each cassette.
2 When you are ready to cast the gels, add the APS and TEMED and mix each gel solution thoroughly. Vary
the amount of TEMED added to control the rate of polymerization. Once these reagents are added,
polymerization begins. You have about 10 min to cast the gradient before the gels begin to solidify at the top.
Work rapidly and carefully.
3 Close the slide valve (out on side of white slide stop button, Fig 5-1). If the outlet tubing is not controlled by a
pump, clamp it off near the gradient former. Add the required volume of the final solution to the reservoir
(back) chamber.
4 Carefully open the slide valve (valve in on button side) and allow just enough solution to flow through the
connector channel to fill it to the edge of the mixing chamber, then close the valve. Be sure no large bubbles
remain to obstruct flow through the channel.
5 Add the required volume of the light solution to the mixing chamber and start the magnetic stirrer.
6 Simultaneously, open the outlet tubing if clamped off, and start the pump. Adjust the pump rate so that the
solution is not forced up in a "fountain" that mixes with the overlying solution.
7 Immediately open the connecting valve between the mixing and the reservoir chamber.
8 Watch the gradient solution enter the caster. It is important that no bubbles disturb the gradient-watch the
delivery carefully.
9 Just as the last of the gradient mix is pumped out of the mixing chamber, add 200 ml of displacement
solution to the mixing chamber and pump it through until almost all of the gradient mix has entered the
cassettes. It is convenient to include a dye in the displacement solution to visually track the boundary
between the gradient mix and the displacement solution.
10 When the gels have reached the desired height and before air is introduced, stop the peristaltic pump. It is
important that no air bubbles disturb the gradient.
reservoir (back)
chamber connector channel
mixing (front)
chamber
slide
stop
slide
valve outlet
connector
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Casting gradient gels 5
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC 17
11 Immediately pipette the water saturated n-butanol onto each gel. Avoid wetting adjacent plastic surfaces
of the caster with n-butanol.
12 Allow the gradient gels to polymerize for 2 hours before disassembling the caster. Gradient gel
polymerization should proceed from the top down. If the peristaltic pump outlet tubing was connected
directly to the caster, it should not be removed until polymerization is complete. Do not leave the water-
saturated n-butanol o the gels overnight as long term exposure of acrylic to butanol will damage the caster.
Fig 5-2. Open and closed positions of slide valve.
open closed
connector
channel
slide
stop
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5 Casting gradient gels
5.3 Pouring gel solutions for gradient gels
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Unloading the gel caster 6
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC 19
6 Unloading the gel caster
WARNING! When using hazardous chemicals, take all suitable protective measures, such as wearing
protective glasses and gloves resistant to the chemicals used. Follow local regulations and instructions for
safe operation and maintenance of the system.
1 Make sure the caster is either near a sink or on a tray so that any liquid leaking out can be contained.
2 Remove the front of the gel caster by loosening and removing the black-knobbed screws and spring clamps.
3 Carefully unload the cassettes from the unit by pulling forward on the separator sheets.
4 Rinse the top surface of each gel with distilled water to remove the n-butanol and any unpolymerized
acrylamide. Remove the separator sheet if still attached and rinse the glass cassettes with water to remove
any acrylamide adhering to the glass plates.
5 Examine the gels for polymerization defects and discard any unsatisfactory gels.
6 Store the acceptable gels in an airtight container at 4 ºC with a small amount of gel storage solution to keep
the gels from drying out.
7 Rinse the gel caster and all tubing with mild detergent then rinse thoroughly with deionized water. Clean the
separator and spacer sheets with a mild detergent and rinse with deionized water.
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6 Unloading the gel caster
20 Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
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