user manual
Hoefer HE99X
Submarine electrophoresis unit
um HE99X-IM/Rev. L0/07-12
Contents
Important Information ...........................................ii
Waste Electrical and
Electronic Equipment (WEEE) ...............................vii
Submarine Electrophoresis Unit
Function and description ........................................1
Specifications........................................................2
Operating instructions ............................................3
Care and maintenance ...........................................8
Troubleshooting .....................................................9
Buffers, volumes, and notes ................................. 10
Loading buffer and sample volumes ......................12
Ordering information ............................................16
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Important Information – English
• If this equipment is used in a manner not specied
by Hoefer, Inc. the protection provided by the
equipment may be impaired.
• This instrument is designed for indoor laboratory
use only.
• Only accessories and parts approved or supplied
by Hoefer, Inc. may be used for operating,
maintaining, and servicing this product.
• Only use a power supply that is CE marked or
safety certied by a nationally recognized testing
laboratory.
• The safety lid must be in place before connecting
the power supply leads to a power supply.
• Turn all power supply controls o and disconnect
the power leads before removing the safety lid.
• Circulate only water or 50/50 water/ethylene glycol
through the heat exchanger if so equipped. Do
not connect the heat exchanger to a water tap or
any coolant source where the water pressure is
unregulated.
• Never introduce antifreeze or any organic solvent
into any part of the instrument. Organic solvents
will cause irreparable damage to the unit!
• Do not operate with buer temperatures above
the maximum specied technical specications.
Overheating will cause irreparable damage to
the unit!
Duležité Informace – Czech
• Pokud by toto zařízení je použito způsobem, který
není podle Hoefer, Inc. ochrana poskytovaná na
základě zařízení může být narušena.
• Tento nástroj je určen pro vnitřní použití v
laboratoři pouze.
• Pouze příslušenství a části schválen, nebo
poskytnutých Hoefer, Inc. mohou být použity pro
provoz, údržbu, a údržbě tohoto výrobku.
• zdroj napájení používají jen že je opatřen
označením CE osvědčena nebo bezpečnost
vnitrostátně uznanými zkušebními laboratoř.
• Bezpečnosti lid musí být zavedena před připojením
napájecí zdroj napájení vede k.
• Turn veškeré napájení kontroly vypnuto a odpojit
před odběrem energie vede bezpečnostní víko.
• Rozeslat pouze voda nebo 50/50 voda/
ethylenglykolu prostřednictvím výměník tepla je
li to vybavena. Nemají připojení výměník tepla s
vodními setřepná nebo jakékoli chladicí kapaliny
zdroje, kde tlak vody je neregulo.
• Nikdy zavést prostředek proti zamrznutí nebo
jakákoli organická rozpouštědla do jakékoli
části z tohoto nástroje. Rozpustidlům způsobí
nenapravitelné poškození jednotka!
• Nejsou provozována s pufru teplotách nad
maximální stanovenou technickými specikacemi.
Přehřátí způsobí nenapravitelné poškození
jednotka!
Vigtig Information – Danish
• Hvis dette udstyr bruges i en måde ikke
speciceret ved Hoefer, Inc. den beskyttelse, som
er blevet forsynet af udstyret kan måske svækkes.
• Dette instrument er designet for indendørs
laboratoriumbrug bare.
• Bare tilbehør og del godkendede eller forsynede
ved Hoefer, Inc. kan måske bruges for drive,
funktionsfejl, og betjening dette produkt.
• bruger Bare en strømforsyning, der er CE
markerede eller sikkerhed, som er blevet attesteret
af en, som nationalt er blevet anerkendt prøve
laboratorium.
• Sikkerhedlåget må være på plads før forbinding
strømforsyningsblyet til en strømforsyning.
• Drejer alle strømforsyningskontroller af og afbryder
kraftblyet før erning sikkerhedlåget.
• Cirkulerer bare vand eller 50/50 vand/ethylene
glykol gennem varmeveksleren i så fald udrustet.
Forbind ikke varmeveksleren til en vandhane
eller nogen kølemiddelkilde hvor vandtrykket er
unregulated.
• Introducerer Aldrig antifreeze eller noget organisk
opløsningsmiddel ind i nogen del af instrumentet.
Organiske opløsningsmidler vil forårsage uboelig
skade til enheden!
• Driver ikke med stødpudetemperaturer
over maksimummet specicerede tekniske
specications. Overheding vil forårsage uboelig
skade til enheden!
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Belangrijke Informatie – Dutch
• Indien deze uitrusting in een manier wordt
gebruikt die niet door Hoefer, Inc. is gespeciceerd
de bescherming die door de uitrusting is verzorgd
kan worden geschaad.
• Dit instrument is voor binnenlaboratoriumgebruik
enkel ontworpen.
• Enkel onderdelen en delen keurden goed of
leverden door Hoefer, Inc. kan voor het bedienen
worden gebruikt, handhavend en onderhouden
van dit product.
• gebruik Enkel een netvoeding die CE is markeerde
of veiligheid die door een is gecerticeerd die
nationaal is herkend testene laboratorium.
• Het veiligheidsdeksel moet in plaats voor het
verbinden van de netvoeding leidt tot een
netvoeding zijn.
• Doe alle netvoedingscontroles Uit en koppel los
de machtleiding voor het verwijderen van het
veiligheidsdeksel.
• Circuleer enkel water of 50/50 water/
ethyleenglycol door de hitte exchanger zo ja
uitrust. Verbind de hitte exchanger naar een
waterkraan of koelmiddelbron niet waar de
waterdruk niet geregulariseerd is.
• Stel Nooit antivriesmiddel of organische
oplosmiddelen in deel van het instrument voor.
Organische oplosmiddelen zullen onherstelbare
schade aan de eenheid veroorzaken!
• Bedien niet met buertemperaturen boven het
maximum speciceerde technische specicaties.
Oververhittend zal onherstelbare schade aan de
eenheid veroorzaken!
Tärkeää Tietoa – Finnish
• Jos tätä varusteita käytetään tavassa ei määritetty
Hoefer, Inc. suojelu ehkäisty varusteille saattaa olla
avuton.
• Tämä väline suunnitellaan sisälaboratoriokäytölle
vain.
• Vain lisävarusteet ja osat hyväksyivät tai toimitti
Hoefer, Inc. oheen ää voi käyttää käyttämiselle,
valvoalle, ja servicing tämä tuote.
• Vain käyttää käyttöjännitettä joka on CE merkitsi
tai turvallisuus joka on todistanut aidoksi ohi
joka on kansallisesti tunnustettnut testaaminen
laboratoriota.
• Turvallisuuskansi täytyy olla paikallaan
ennen yhdistäminen käyttöjännitelyijyjä
käyttöjännitteeseen.
• Kiertää kaikki käyttöjännitevalvonnat ja irrottaa
valtalyijyt ennen poistaminen turvallisuuskantta.
• Kiertää vain vesi tai 50/50 vesi/ethyleneä glycol
siinä tapauksessa varustetun lämmönvaihtimen
läpi. Älä yhdistä lämmönvaihdinta
vesinapautukseen eikä jäähdytysnestelähteeseen,
missä vesipaine on unregulated.
• Pakkasneste eikä orgaaninen liuotin välineen
osassa ei esitele Koskaan. Orgaaniset liuottimet
aiheuttavat korvaamattoman vahingon yksikköön!
• Ei käytä puskuria yllä olevia lämpötiloja
enintään määritetyillä teknisillä täsmennyksillä.
Ylikuumeneminen aiheuttaa korvaamattoman
vahingon yksikköön!
Information Importante – French
• Si cet équipement est utilisé dans une manière pas
spécié par Hoefer, Inc. la protection fourni par
l’équipement pourrait être diminuée.
• Cet instrument est conçu pour l’usage de
laboratoire intérieur seulement.
• Seulement les accessoires et les parties ont
approuvé ou ont fourni par Hoefer, Inc. pourrait
être utilisé pour fonctionner, maintenir, et
entretenir ce produit.
• utilise Seulement une alimentation qui est
CET a marqué ou la sécurité certié par un
nationalement reconnu essayant le laboratoire.
• Le couvercle de sécurité doit être à sa place avant
connecter l’alimentation mene à une alimentation.
• Tourner tous contrôles d’alimentation de et
débrancher les avances de pouvoir avant enlever le
couvercle de sécurité.
• Circuler seulement de l’eau ou 50/50 glycol d’eau/
éthylène par l’exchanger de chaleur si si équipé. Ne
pas connecter l’exchanger de chaleur à un robinet
d’eau ou à la source d’agent de refroidissement où
la pression d’eau est non régulée.
• Ne Jamais introduire d’antigel ou du dissolvant
organique dans n’importe quelle partie de
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•
l’instrument. Les dissolvants organiques causeront
des dommages irréparables à l’unité!
• Ne pas fonctionner avec les températures de
tampon au-dessus du maximum a spécié des
spécications techniques. La surchaue causera
des dommages irréparables à l’unité !
Wichtige Informationen – German
• Wenn diese Ausrüstung gewissermaßen nicht
angegeben durch Hoefer, Inc. verwendet wird,
kann der durch die Ausrüstung zur Verfügung
gestellte Schutz verschlechtert werden.
• Dieses Instrument wird für den
Innenlaborgebrauch nur dafür entworfen.
• Nur Zusätze und Teile genehmigten oder lieferten
durch Hoefer, Inc. kann für das Funktionieren,
das Aufrechterhalten, und die Wartung dieses
Produktes verwendet werden.
• Verwenden Sie nur eine Energieversorgung,
die CE gekennzeichnet oder durch ein national
anerkanntes Probelaboratorium bescheinigte
Sicherheit ist.
• Der Sicherheitsdeckel muss im Platz vor dem
Anschließen der Energieversorgung sein führt zu
einer Energieversorgung.
• Alle Energieversorgungssteuerungen abdrehen
und die Macht trennen führt vor dem Entfernen
des Sicherheitsdeckels.
• Nur Wasser oder 50/50 Glykol des Wassers/
Äthylens durch den Wärmeaustauscher, wenn so
ausgestattet, in Umlauf setzen. Verbinden Sie den
Wärmeaustauscher mit einem Wasserklaps oder
jeder Kühlmittel-Quelle nicht, wo der Wasserdruck
ungeregelt wird.
• Führen Sie nie Frostschutzmittel oder jedes
organische Lösungsmittel in jeden Teil des
Instrumentes ein. Organische Lösungsmittel
werden nicht wiedergutzumachenden Schaden
der Einheit verursachen!
• Mit Puertemperaturen über angegebenen
technischen Spezizierungen des Maximums
nicht funktionieren. Die Überhitzung wird nicht
wiedergutzumachenden Schaden der Einheit
verursachen!
Informazioni Importanti – Italian
• Se quest’apparecchiatura è usata in un modo
specicato da Hoefer, Inc. la protezione fornito
dall’apparecchiatura potrebbe essere indebolita.
• Questo strumento è disegnato per l’uso di
laboratorio interno solo.
• Solo gli accessori e le parti hanno approvato o
hanno fornito da Hoefer, Inc. potrebbe essere
usato per operare, per mantenere, e per revisionare
questo prodotto.
• usa Solo un alimentatore che è CE ha marcato
o la sicurezza certicato da un nazionalmente
riconosciuto testando il laboratorio.
• Il coperchio di sicurezza deve essere nel luogo
prima di collegare i piombi di alimentatore a un
alimentatore.
• Spegne tutto i controlli di alimentatore e
disinserisce i piombi di potere prima di togliere il
coperchio di sicurezza.
• Circola solo l’acqua o 50/50 glicole di acqua/
etilene attraverso lo scambiatore di calore se
così equipaggiato. Non collegare lo scambiatore
di calore a un rubinetto di acqua o qualunque
fonte di refrigerante dove la pressione di acqua è
sregolata.
• Non introduce mai l’antigelo o qualunque solvente
organico in qualunque parte dello strumento. I
solventi organici causeranno il danno irreparabile
all’unità!
• Non opera con le temperature di tampone al di
sopra del massimo ha specicato le descrizioni
tecniche. Il surriscaldamento causerà il danno
irreparabile all’unità!
Viktig Informasjon – Norwegian
• Hvis dette utstyret blir brukt i en måte ikke
spesisert ved Hoefer, Inc. beskyttelsen som ha
blitt git av utstyret kan bli svekket.
• Dette instrumentet er utformet for innendørs
laboratoriumbruk bare.
• Bare tilbehør og deler godkjente eller forsynte ved
Hoefer, Inc. kan bli brukt for drive, vedlikeholde, og
betjene dette produktet.
• bruker Bare en kraftforsyning som er CE merket
eller sikkerhet som ha blitt sertisert av et som
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•
nasjonalt ha blitt anerkjent prøver laboratorium.
• Sikkerheten lokket må være på plass før forbinding
kraftforsyningene blyene til en kraftforsyning.
• Vender all kraftforsyningsstyring av og frakopler
kreftene blyene før erning sikkerheten lokket.
• Sirkulerer bare vann eller 50/50 vann/ethylene
glykol gjennom oppvarmingen veksleren i så fall
utstyrer. Ikke forbind oppvarmingen veksleren
til en vanntapp eller noe kjølemiddelkilde hvor
vannet trykket er unregulated.
• Introduserer Aldri antifreeze eller noe organisk
løsemiddel inn i noe del av instrumentet.
Organiske løsemiddler vil forårsake irreparabel
skade på enheten !
• Driver med buertemperaturer over maksimum
ikke spesiserte teknisk spesikasjoner. Å
overoppheting vil forårsake irreparabel skade på
enheten !
Wazne Informacje – Polish
• Jeżeli ten sprzęt jest wykorz ystywany w sposób
nie określone przez Hoefer, Inc. do ochrony
przewidzianej przez urządzenie może zostać
obniżony.
• Instrument ten jest przeznaczony do użytku w
laboratoriach kryty tylko.
• Tylko akcesoriów i części zatwierdzone lub
dostarczone przez Hoefer, Inc. mogą być
wykorzystane do eksploatacji, utrzymania i obsługi
tego produktu.
• korzystać jedynie zasilacza że jest noszące
oznakowanie CE lub bezpieczeństwa
uwierzytelnione przez uznane na poziomie
krajowym laboratorium badawcze.
• Bezpieczeństwo lid musi być w miejsce przed
podłączeniem zasilania prowadzi do zasilania.
• Zaś wszystkie źródła zasilania urządzenia sterujące
o i odłączyć moc prowadzi przed odbiorem
bezpieczeństwa lid.
• Krążą tylko wody lub wody 50/50/ethylene glycol
wymiennik ciepła poprzez jeśli tak wyposażone.
Nie należy połączyć wymiennik ciepła woda z
kranu lub jakimkolwiek chłodziwo źródła, jeżeli
ciśnienie wody jest nieuregulowanych.
• Nigdy nie wprowadzać rozpuszczalnika
organicznego przeciw zamarzaniu lub
jakichkolwiek na dowolną część dokumentu.
Rozpuszczalniki organiczne spowoduje
nieodwracalne szkody dla jednostki!
• Nie działają w buforze temperatury powyżej
maksymalnego określone specykacje techniczne.
Przegrzania spowoduje nieodwracalne szkody dla
jednostki!
Informações Importantes –
Portuguese
• Se este equipamento é usado numa maneira
não especicada por Hoefer, Inc. que a
protecção fornecida pelo equipamento pode ser
comprometida.
• Este instrumento é projectado para uso de interior
de laboratório só.
• Só acessórios e partes aprovaram ou forneceu por
Hoefer, Inc. pode ser usada para operar, manter, e
servicing este produto.
• Só usa um estoque de poder que é CE marcou
ou segurança registrada por um nacionalmente
reconhecido testando laboratório.
• A tampa de segurança deve estar em lugar antes
de ligar o estoque de poder leva a um estoque de
poder.
• Desliga todos controlos de estoque de poder e
desconecta os chumbos de poder antes de retirar a
tampa de segurança.
• Circulam só água ou 50/50 glicol de água/ethylene
pelo exchanger de calor se for assim equiparam.
Não ligue o exchanger de calor a uma torneira de
água nem qualquer fonte de refrigerante onde a
pressão de água é não regulado.
• Nunca introduz anticongelante nem qualquer
orgânico solvente em qualquer parte do
instrumento. Orgânico solvente causará agressão
irreparável à unidade!
• Não opera com temperaturas de buer acima
do máximo especicou especicações técnicas.
Superaquecer causará agressão irreparável à
unidade!
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•
Información Importante – Spanish
• Si este equipo es utilizado en una manera no
especicado por Hoefer, Inc. la protección
proporcionado por el equipo puede ser dañada.
• Este instrumento es diseñado para el uso interior
del laboratorio sólo.
• Sólo accesorios y partes aprobaron o suministraron
por Hoefer, Inc. puede ser utilizado para operar,
para mantener, y para atender a este producto.
• Sólo utiliza una alimentación que es CE marcó o
la seguridad certicada por un nacionalmente
reconocido probando el laboratorio.
• La tapa de la seguridad debe estar en el lugar
antes de conectar la alimentación lleva a una
alimentación.
• Apaga todos controles de alimentación y
desconecta los plomos del poder antes de quitar la
tapa de la seguridad.
• Circula sólo agua o 50/50 glicol de agua/etileno
por el intercambiador de calor si ése es el caso
equiparon. No conecte el intercambiador de calor a
un toque de la agua ni cualquier fuente del líquido
refrigerante donde la presión del agua está libre.
• Nunca introduce anticongelante ni algún solvente
orgánico en cualquier parte del instrumento. Los
solventes orgánicos causarán daño irreparable a
la unidad!
• No opera con temperaturas de búfer encima del
máximo especicó especicaciones técnicas.
Recalentar causará daño irreparable a la unidad!
• Säkerheten locket måste vara på platsen före
koppla kraften tillgången blyen till en kraft tillgång.
• Vänder sig alla kraft tillgång kontroller av och
kopplar bort kraften blyen före ytta säkerheten
locket.
• Cirkulerar bara vatten eller 50/50 vatten/ethylene
glycol genom värmen exchanger i så utrustad fall.
Inte kopplar värmen exchanger till en vatten kran
eller något kylmedel källa där vattnet trycket är
unregulated.
• Inför aldrig kylvätska eller något organiska
lösningsmedel in i någon del av instrumentet.
Organiskt lösningsmedel ska orsaka irreparable
skada till enheten!
• Använd inte med buert temperaturer över
det högsta angivna tekniska specikationerna.
Överhettning skulle orsaka irreparabla skador
på enheten!
Viktig Information – Swedish
• om denna utrustning används i ett sätt som
inte har speciceras av Hoefer, Inc. skyddet
tillhandahöll vid utrustningen kan skadas.
• Detta instrument formges för
inomhuslaboratorium användning bara.
• Bara medhjälpare och delar godkände eller
levererade vid Hoefer, Inc. kan användas för
fungera, underhålla, och servicing denna produkt.
• använder bara en kraft tillgång som är CE
markerade eller säkerhet intygade vid en nationellt
erkänd testande laboratorium.
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Waste Electrical and Electronic Equipment (WEEE)
English
French
German
Italian
Spanish
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.
Ce symbole indique que les déchets relatifs à l’équipement
électrique et électronique ne doivent pas être jetés comme
les ordures ménagères non-triées et doivent être collectés
séparément. Contactez un représentant agréé du fabricant
pour obtenir des informations sur la mise au rebut de
votre équipement.
Dieses Symbol kennzeichnet elektrische und elektronische
Geräte, die nicht mit dem gewöhnlichen, unsortierten
Hausmüll entsorgt werden dürfen, sondern separat
behandelt werden müssen. Bitte nehmen Sie Kontakt mit
einem autorisierten Beauftragten des Herstellers auf, um
Informationen hinsichtlich der Entsorgung Ihres Gerätes
zu erhalten.
Questo simbolo indica che i rifiuti derivanti da
apparecchiature elettriche ed elettroniche non devono essere
smaltiti come rifiuti municipali indifferenziati e devono invece
essere raccolti separatamente. Per informazioni relative alle
modalità di smantellamento delle apparecchiature fuori uso,
contattare un rappresentante autorizzato del fabbricante.
Este símbolo indica que el equipo eléctrico y electrónico no
debe tirarse con los desechos domésticos y debe tratarse por
separado. Contacte con el representante local del fabricante
para obtener más información sobre la forma de desechar
el equipo.
Swedish
Denna symbol anger att elektriska och elektroniska
utrustningar inte får avyttras som osorterat hushållsavfall och
måste samlas in separat. Var god kontakta en auktoriserad
tillverkarrepresentant för information angående avyttring
av utrustningen.
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Submarine Electrophoresis Unit Function and description
The Hoefer® HE99X unit electrophoretically
separates nucleic acid fragments in a submarine
gel. The gel is first cast in a gel caster, which is
available in three lengths. Once the gel sets, the
running tray is transferred to the platform of
the electrophoresis unit and the gel is submerged
under running buffer.
Fig 1. Horizontal submarine unit
main components.
Gel casting kits, combs and comb
backs may be ordered separately;
Fig 2 illustrates a casting kit, and
the ordering section tabulates all
comb sizes and accessories.
Rest thumbs
on both posts
(protruding
through each
end of lid) while
lifting both tabs
on lid for easy
lid removal.
running
platform
Color-coded leads connect
electrodes in the unit base
to the power supply.
spirit
level
buffer
chamber
electrode
post (2)
leveling
feet (4)
p1
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This declaration of conformity is
only valid for the instrument when
it is:
• used in laboratory locations,
• used as delivered from Hoefer,
Inc. except for alterations
described in the user manual,
and
• connected to other CE-labeled
instruments or products
recommended or approved by
Hoefer, Inc.
Unpacking
Unwrap all packages carefully and compare
contents with the packing list, making sure all
items arrived. If any part is missing, contact
your local Hoefer, Inc. 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.
Specifications
Max. voltage 200 V
Max. wattage 20 W
Max. amperage 100 mA
Max. operating temp. 45 °C
Max. buffer volume 1.2 liters
Gel size 15 cm wide × 10, 15,
or 20 cm long
Environmental
operating conditions Indoor use: 4–40 °C
Humidity up to 80%
Altitude up to 2000 m
Installation category II
Pollution degree 2
Dimensions (w × l × d) 18.2 × 36 × 14 cm
(includes electrode posts) (7.2 × 14.2 × 5.5 in.)
Weight 0.82 kg (1.8 lb)
(base, lid, and leads only)
Product certifications EN61010–1, UL61010A-1,
CSA C22.2 1010.1,
CE Certified
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Operating instructions
Before you start…
1. Wash all components
with a dilute solution of
laboratory detergent and rinse
thoroughly.
2. Level the unit by placing the
spirit level on the running
platform and adjusting the
leveling feet.
Volume for 3-mm thick gels
tray size (cm) agarose (ml)
15 × 10 45
15 × 15 68
15 × 20 90
Caution! Ethidium bromide is
a known mutagen. Always wear
gloves when handling.
Agarose gels are first cast in the gel casting kit,
and samples are then loaded into the wells and
electrophoretically separated. The fluorescent
dye ethidium bromide can be added to the gel
or electrophoresis buffer or both in order to
track separation progress. At the completion
of electrophoresis, the gel may be stained and
photographed, blot transferred, or dried for
autoradiography.
Casting the gel
Prepare the solutions
1
Prepare about 1.3 liters of running buffer. Up to 100 ml
of buffer is required for the gel and 1.2 liters for the
buffer chamber. Refer to page 10 for recipes of three
commonly used electrophoretic running buffers.
2
Prepare the sample loading buffer. Refer to page 12
for a recipe and tabulated volume capacity for each
comb size.
3
Prepare agarose solution(s).
Dissolve agarose in running buffer, heat according to
instructions accompanying the agarose, and allow the
solution to cool to 50 °C before pouring into the
running tray.
Optional: Add 0.5 µg/ml ethidium bromide to the gel
solution in order to facilitate observation of separation
progress during electrophoresis.
p3
•
Prepare the casting tray and pour the gel
1
Install a foam pad at each end of the casting tray.
Use a comb as a placement guide so that the pad
~
adheres
the comb into the bottom of the tray, oriented so
that it fits completely across the tray along the side
that is 16 cm wide. Peel off the adhesive backing on
the foam pad, align the pad on the comb, adhesive
side toward the inside wall of the tray, and slide the
comb against the wall. Press the foam pad in place
and repeat with second pad on the wall opposite the
first pad.
1 mm from the bottom of the tray: Lay
~
Fig 2. Gel casting kit.
Running tray installation: Approach
the foam pad with one end of the
running tray (Arrow A) and then
gently press the tray edge against
the pad, compressing it enough
to allow the opposite end of the
running tray to drop fully into
the casting tray (Arrow B) before
sealing against the foam pad.
Note: Grooves in the running tray
create ridges at both ends of the
gel to prevent it from slipping
or floating. If these ridges are
not desired, either tape over the
grooves before casting, or trim off
ridges with a spatula after the run.
UVT gel running
tray (15 × 20 cm)
B
foam
pads (2)
Color-coded dots (2)
on handles orient the
running tray on the
running platform.
•
The comb back
fits into any set
of slots. the comb
back assembly
shown in Fig 3 is
oriented to fit into
this slot.
A
gel casting tray
(15 × 20 cm)
p4
•
Fig 3. Assembled comb.
comb back
screws
2
Seat the running tray between the foam pads in the
casting tray by placing one end of the tray against
the foam pad, slightly compressing it, then seating
the other end of the tray against the opposite foam
pad. (See arrows A and B in Fig 2.) The running tray
should lay flush against the bottom of the casting tray.
3
Place the casting tray assembly on a leveling surface
and level, using the spirit level on the running tray as
a guide. Check that the comb assembly leaves
of space between the comb bottom and the running
tray. Remove the level and the comb assembly.
~
1 mm
~
Prepare the combs
1
Align the two slots in the comb with the loosened
thumb screws of the comb back. Tighten the screws
until the comb is just supported.
2
Place the comb assembly into a set of slots on the
running tray seated in the casting tray. Adjust the
comb so that the bottom of the teeth are
from the running tray. Tighten the screws to secure
the comb.
To run twice as many samples on the 15 and 20 cm
trays, prepare two comb assemblies and place one
near the cathode end, indicated by the black dot, and
one at the center.
~
1.0 mm
~
p5
•
Final casting steps
1
Pour the agarose solution (cooled to 50 °C) onto the
running tray seated in the casting tray. Orient the
comb assembly so that it is at the end of the tray
opposite the direction of migration (typically at the
cathode [–] end, which is marked by a black dot on
the handle). Fit the comb assembly into the slots.
2
Allow a minimum of 30 min for the gel to set, then
remove the comb carefully: partially lift and slightly
tilt the comb at one end and slowly withdraw it from
the gel. (Pulling the comb straight up creates a
vacuum in the wells that may lift the gel out of
the tray.)
3
Lift the running tray out of the casting tray and
transfer it with the gel to the horizontal unit. Orient
the running platform so that the sample will “run to
red”. That is, place the sample wells at the cathode
(–) end, which is indicated by a black dot. A notch on
either side of the running tray centers the tray on the
running platform.
p6
•
Caution! Wear UV safety goggles
and protect skin when using a
UV lamp.
Note: Refer to the Buffers,
volumes, and notes section
for additional information and
guidelines on page 10.
See page 12 for a sample
loading buffer recipe and well
volumes for various comb sizes
in gels of different thicknesses.
Important! If running two sets
of samples in one gel, monitor
the run closely and stop
electrophoresis when the marker
dye approaches the wells in
the center.
Preparing for electrophoresis
1
Optional: To monitor separation progress, either add
0.5 µg/ml (final conc.) of ethidium bromide to the
running buffer now or add 50 µg/ml (final conc.)
ethidium bromide to the sample buffer. To visualize
progress, turn off the power supply, remove the lid
assembly, and hold a portable UV lamp near the gel.
Note: Adding ethidium bromide to the running or
sample buffer slows migration slightly. Detection by
this method is not as sensitive as by staining after the
electrophoresis run. See the DNA detection section,
for more details (page 14).
2
Fill the chamber with buffer until the gel is
submerged
~
1 mm.
~
3
Load the samples. Add the sample to 1/5 volume of
the sample loading buffer. Mix each sample and load
into a well with a micro-pipet, taking care to avoid
puncturing the well bottom or entrapping bubbles.
4
Place the lid on the unit so that the cathode (black
lead) is at the end nearest the samples. (Nucleic acid
samples migrate toward the anode.)
5
Connect the color-coded leads (red to red, and black
to black) to an approved power supply. Set the voltage
and timer (if available). Agarose gels are typically
run at constant voltage under a voltage gradient in
the range of 2–5 V/cm. The distance between the
electrodes is
a gradient of 5 V/cm.
~
26 cm, so a setting of 130 V results in
~
p7
•
After electrophoresis
1
Important! Always turn off the power supply and
disconnect the leads before removing the lid.
2
If no ethidium bromide was added to the gel or
sample before the run, stain the gel now in a solution
of 0.5 to 1.0 µg/ml ethidium bromide in water
or buffer.
3
Clean the unit as described in the next section.
Care and maintenance
Cleaning
• Never autoclave or heat any component
above 45 °C.
• Never use abrasive cleansers.
• Do not expose the unit to solutions or vapors
of aromatic or halogenated hydrocarbons,
ketones, esters, alcohols (over 30%), or
concentrated acids (over 25%).
The unit is resistant to all common electrophoresis buffers, but we recommend a thorough washing with a mild detergent after each use. Rinse
with distilled water and allow to air dry.
To remove DNase and RNase contamination,
fill the unit with 3% hydrogen peroxide (H2O2),
soak for 10 minutes, then rinse thoroughly with
DEPC-treated, autoclaved, deionized water.
(Sambrook and Russell, et al. 1:7.82)
p8
•
Troubleshooting
problem solution
Sample well deformed Allow the gel to set for a minimum of 1 hour and make
Remove the comb at a slight angle and very slowly to
Take care to not damage the well with the pipet while
Samples not running If the comb is warped, replace.
along a straight path
Circulate buffer if it becomes depleted by stopping
Double-banded pattern Make sure the comb remains vertical after the gel is cast
Decrease the buffer level to 1 mm above the top of the
Poor band resolution Add Ficoll™, glycerol, or sucrose to the sample loading
Make sure the sample is completely dissolved.
Reduce the sample concentration.
Reduce the sample volume.
Reduce voltage to 5 V/cm.
Be sure the well floor is at least 1 mm thick to prevent
Reduce the salt concentration of the sample.
Check enzyme activity; the sample may require longer
Prepare fresh sample if you suspect nuclease
Choose agarose with a low endosmosis value.
Foam pads peel off Do not press the running tray into place. Install as
If the running tray is warped, replace.
sure it is at room temperature before removing the comb.
prevent the gel from breaking.
loading the sample; aim for the center of the well and do
not puncture the bottom with the pipet tip.
(Cool agarose to 50 °C to prevent the tray from warping.)
the run and pipetting the buffer from one chamber to
the other.
so that the well shape is not distorted.
gel in order to reduce the temperature gradient in the gel.
buffer to ensure that the sample sinks to the bottom of
the well. (Ficoll is the recommended agent.)
samples from leaking through the bottom.
digestion or a different restriction buffer.
contamination.
described on page 4.
p9
•
Important! Do not adjust the
pH of these buffers once
they are prepared according
to the recipe!
Notes, buffers, and volumes
Running buffers for DNA in agarose gels
Recipes for the three most commonly used
running buffers for DNA electrophoresis are
listed below. The buffering capacity of both
TBE and TPE is usually sufficient so that buffer
circulation is unnecessary. Circulation may be
required during runs longer than 3 hours or
when using the TAE buffer.
1. 10X Tris-borate-EDTA (TBE) stock buffer
(0.89 M Tris, 0.89 M boric acid, 20 mM EDTA,
pH ≈8.3, 1000 ml)
Tris base (FW 121.1) 0.89 M 108.0 g
Boric acid (FW 61.8) 0.89 M 55.0 g
EDTA solution 0.02 M 40.0 ml
(0.5 M, pH 8.0, solution 4)
Deionized H2O to 1000.0 ml
Stir. Do not adjust pH.
Before use dilute either to:
0.5X, to yield 45 mM Tris base, 45 mM boric acid,
and 1 mM EDTA. This dilution is often used because
current remains low, resulting in less heat.
—or—
1X, to yield 89 mM Tris base, 89 mM boric acid, and
2 mM EDTA.
a
•
p10
2. 10X Tris-phosphate-EDTA (TPE) stock buffer
a
(0.89 M Tris, 0.89 M phosphoric acid, 20 mM EDTA,
pH ≈8.1, 1000 ml)
Tris base (FW 121.1) 0.89 M 108.0 g
Phosphoric acid (85%) 0.23 M 15.5 ml
EDTA solution 0.02 M 40.0 ml
(0.5 M, pH 8.0, solution 4)
Deionized H2O to 1000.0 ml
Stir. Do not adjust pH. Dilute to 1X, to yield 89 mM
Tris base, 23 mM phosphoric acid, and 2 mM EDTA.
3. 10X Tris-acetate-EDTA (TAE) stock buffer
a
(0.4 M Tris, 0.2 M acetic acid, 10 mM EDTA,
pH ≈8.4, 1000 ml)
Tris base (FW 121.1) 0.40 M 48.4 g
Acetic acid (99.5%) 0.20 M 11.4 ml
EDTA solution
(0.5 M, pH 8.0, solution 4) 0.01 M 20.0 ml
Deionized H2O to 1000.0 ml
Stir. Do not adjust pH. Dilute to 1X before use to
yield 40 mM Tris base, 20 mM acetic acid,
and 1 mM EDTA.
4. EDTA solution
(ethylenediamine tetraacetic acid)
b
(0.5 M, pH 8.0, 100 ml)
Na2EDTA·2H2O, (FW 372.2) 0.5 M 18.6 g
Deionized H2O to 70.0 ml
NaOH (10 M) to pH 8.0 ≈5.0 ml
Deionized H2O to 100.0 ml
a
Sambrook J. and Russell, D.W., (2001) Molecular Cloning: A Labora-
tory Manual, A1.17
b
Current Protocols in Molecular Biology (1993) A.2.1
•
p11
Loading buffer and sample volumes
Loading buffer
(5X, 25% Ficoll 400, 0.25% Bromphenol blue†, 10 ml)
Deionized H2O to 7.0 ml
Ficoll 400 2.5 g
Bromophenol blue (FW 691.9) 25.0 mg
Deionized H2O to 10.0 ml
Add 1 volume loading buffer to 4 volumes of sample.
(Loading buffer increases solution density.)
Note 1: Sucrose or glycerol may be used instead of
Ficoll 400.
Note 2: Xylene cyanol (0.25%), which migrates
more slowly than bromophenol blue, can be
added as an additional marker if desired. The
agarose concentration determines the position
of the dye bands relative to a polynucleotide.
†
Tracking dyes may be omitted to eliminate obscuring or dragging
effects caused by comigration with smaller nucleic acids.
Comb specifications and well volumes
well well sample vol.
code no. of width thickness per 1 mm
number wells (mm) (mm) depth (µl)
HE91A-P-1.5 1/2 113/10 1.5 171/14.5*
HE91A-P-3.0 1/2 113/10 3.0 342/29.0*
HE91A-10-1.5 10 9.7 1.5 14.5
HE91A-10-3.0 10 9.7 3.0 30.0
HE91A-15-1.0 15 7.1 1.0 7.1
HE91A-15-1.5 15 7.1 1.5 10.6
HE91A-15-3.0 15 7.1 3.0 21.3
HE91A-20-1.0 20 4.7 1.0 4.7
HE91A-20-1.5 20 4.7 1.5 7.1
HE91A-20-3.0 20 4.7 3.0 14.2
HE91A-30-1.0 30 3.0 1.0 3.0
* Preparative combs form two reference wells (for MW standards), one on
each side of the preparative well. The first number is sample volume/
mm depth in the preparative well; the second is volume/mm in the
reference well.
•
p12
Agarose gel electrophoresis notes
Agarose gel electrophoresis can be used to
separate DNA fragments down to 0.1 kb or
less. Polyacrylamide gels are typically used for
fragments smaller than 1 kb.
DNA mobility
The suggested agarose concentration for
separating fragments of various sizes is listed
below. Other factors affecting separation results
include the selected running buffer, the voltage
setting, the temperature, and the presence of
ethidium bromide.
Agarose concentrations for separating
DNA fragments of various sizes
effective range of resolution
agarose (%) of linear DNA fragments (kb)
0.5 1 to 30
0.7 0.8 to 12
1.0 0.5 to 10
1.2 0.4 to 7
1.5 0.2 to 3
†
Current Protocols in Molecular Biology, p 2.5.2 (1993).
†
A common standard is a Hind III digest of
lambda phage, which gives eight fragments ranging in size from 0.1 to 23 kb. The bands are well
resolved when run 2 hours on a 20 cm long 1%
agarose gel in 0.5X TBE buffer at 150 V.
p13
•
Note: For an example of
RNA electrophoresis, refer to
Molecular Cloning: A Laboratory
Manual by J. Sambrook and
D.W. Russell.
Caution! Ethidium bromide is
a known mutagen. Always wear
gloves when handling.
Caution! Wear UV safety goggles
and protect skin when using any
UV light source.
Note: Ethidium bromide slows
DNA migration by
Note: Minimize the staining time
to prevent small nucleic acid
fragments from diffusing out of
the gel.
~
15%.
~
RNA mobility
RNA can also be separated on the basis of
size. To avoid irregularities due to secondary
structure, RNA is denatured either before or
during electrophoresis. For example, RNA
fragments previously denatured with glyoxal
and dimethylsulfoxide can be separated on
neutral agarose gels, or RNA can be fractionated
on agarose gels containing methylmercuric
hydroxide or formaldehyde.
RNA samples usually require longer runs or
buffers that are easily depleted, and so require
circulation. The Hoefer SUB20C and SUB25C
horizontal units are recommended for this application rather than the HE33.
DNA detection
DNA can be detected either by the fluorescence
of bound ethidium bromide or by autoradiography of radio-labeled DNA.
Ethidium bromide (0.5 µg/ml) can be added
to running buffer to monitor sample progress
because the dye’s fluorescence reveals DNA
under UV light. (To check band location, turn
off the power supply and remove the lid of the
agarose unit. Hold a portable UV lamp near the
running tray. Replace the lid and turn on the
power again to resume electrophoresis.)
Alternatively, after electrophoresis, stain the
gel in an ethidium bromide solution (0.5 µg/
ml H2O) for 15 to 60 minutes and then
view or photograph the sample on a UV
transilluminator.
To photograph the gel, either place the running
tray on the transilluminator surface or slide the
gel onto the surface for maximum exposure. The
running tray is 95% transparent to 302 nm light
and 40% transparent to 254 nm light. If you
•
p14
place the gel on the transilluminator, ensure that
it lies flat by cutting off the ridges formed by the
grooves in the running tray. (Do not damage the
transilluminator surface; trim both ends of the
gel with a spatula while it is still in the tray, lift
away the ridges, and then slide the gel onto the
transilluminator.) For viewing, 302 nm light is
recommended for both acceptable sensitivity and
reduced photonicking.
To reduce the background fluorescence of
unbound ethidium bromide, the gel can be
destained by soaking it for 5 minutes in 0.01
M MgCl2, or for 1 hour in 0.001 M MgSO4.
Destaining makes it easier to detect small quantities (less than 10 ng) of DNA. (Sambrook and
Russell, A9.4.).
Transfer
Before transfer, trim off the ridges at both
ends of the gel to ensure even gel contact with
the membrane.
Bibliography
Ausubel, et al., (eds). Current Protocols in
Molecular Biology. Greene Publishing and
Wiley-Interscience. New York (1993).
Sambrook, J., and Russell, D.W., Molecular
Cloning: A Laboratory Manual. Cold Spring
Harbor Laboratory Press. (2001).
•
p15
Ordering information
product quantity code number
HE99X Horizontal Agarose Submarine Unit, complete. 1 HE99X-15-1.5
Includes basic unit, 15 × 20 cm gel casting kit,
one 1.5 mm-thick 15-well comb and comb back.
HE99X Horizontal Agarose Submarine Unit, basic. 1 HE99X
Includes spirit level. (Order gel casting kit,
comb and comb back separately.)
Accessories and replacement parts
Buffer chamber assembly only 1 HE90X
Comb back for HE99X comb with 2 screws 1 HE91-BK
Lid with power cables 1 HE96X
High voltage leads, set 1 SE6056-HV
Mylar sealing tape (1 roll, 66 mm) 1 SE1510
Foam sealing gaskets 4 HE98X
Leveling feet 4 HE99XRK-1
Companion products
MacroVue™ UV-20 Transilluminator 115 V~ 1 UV20-115V
MacroVue™ UV-20 Transilluminator 230 V~ 1 UV20-230V
HE99X gel casting kits
Each size includes 1 gel casting tray, 1 UVT running tray, and 4 foam sealing gaskets
15 × 10 cm 1 HE97X-10
15 × 15 cm 1 HE97X-15
15 × 20 cm 1 HE97X-20
HE99X gel casting trays
15 × 10 cm 1 HE95X-10
15 × 15 cm 1 HE95X-15
15 × 20 cm 1 HE95X-20
HE99X gel running trays
15 × 10 cm 1 HE92X-10
15 × 15 cm 1 HE92X-15
15 × 20 cm 1 HE92X-20
p16
•
Combs
comb thickness well width
no. of wells (mm) (mm) code number
1/2* 1.5 113/10 HE91A-P-1.5
1/2* 3.0 113/10 HE91A-P-3.0
10 1.5 9.7 HE91A-10-1.5
10 3.0 9.7 HE91A-10-3.0
15 1.0 7.1 HE91A-15-1.0
15 1.5 7.1 HE91A-15-1.5
15 3.0 7.1 HE91A-15-3.0
20 1.0 4.7 HE91A-20-1.0
20 1.5 4.7 HE91A-20-1.5
20 3.0 4.7 HE91A-20-3.0
30 1.0 3.0 HE91A-30-1.0
* Preparative combs form two marker wells, one on each side of the preparative well. The first value in each column refers
to the prep well, the second to the reference well.
HE90X
buffer chamber
SER11
spirit level
HE96X
lid w/power cables
SE6056-HV
high voltage
leads, set
HE99XRK-1
leveling foot
HE91-BK
comb back with
2 screws
combs
(see list for sizes)
HE31-S
screws
•
p17
Hoefer, Inc.
84 October Hill Road
Holliston, MA 01746
Toll Free: 1-800-227-4750
Phone: 1-508-893-8999
Fax: 1-508-893-0176
E-mail: support@hoeferinc.com
Web: www.hoeferinc.com
Hoefer is a registered trademark
of Hoefer, Inc.
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All rights reserved.
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