INSTRUCTION MANUAL
Semi-Dry Blotting Systems
EBU-3000
EBU-4000
EBU-6000
www.cbsscientific.com 2 Semi-Dry Blotter Manual 12/12
TABLE OF CONTENTS
Page
Important User Information 3-4
Section 1 General Information
1.1 Introduction 5
1.2 Specifications 5
1.3 Safety 5
Section 2 Description of parts
2.1 Unpacking EBU-3000, 4000 or 6000 6
2.2 Components/Assembly 6
Section 3 Instructions for Use
3.1 Blotting Unit Preparation 7-11
Section 4 References 12
Section 5 Maintenance of Equipment
5.1 Care and Handling 13
5.2 Maintenance 13
Section 6 Equipment and Accessories 14
www.cbsscientific.com 3 Semi-Dry Blotter Manual 12/12
IMPORTANT USER INFORMATION
This Instruction Manual will explain how to use this product safely and effectively. Please read and
carefully follow the instruction manual in its entirety.
The triangle/exclamation mark symbol alerts the user of the product to important
operational, maintenance, and/or warranty requirements.
The triangle/lighting bolt symbol alerts the user of the product to potentially hazardous
electrical exposure.
Failure to adhere to the instructions could result in personal and/or laboratory hazards, as well as
invalidate any warranty. Always turn off the DC power source prior to disconnecting power cords
from the product. Disconnect power cords from the power source first and then from the product.
For maximum safety, always operate this system in an isolated, low traffic area, not accessible to
unauthorized personnel. Never operate damaged or leaking equipment.
WARRANTY AND LIABILITY
This product was produced utilizing the highest practical standards of materials, workmanship, and
design. C.B.S. Scientific warrants that the product has been tested and will meet or exceed published
specifications. This warranty is valid only if the product has been operated and maintained according to
the instructions provided.
C.B.S. Scientific warrants this product to be free from defects in materials and workmanship under normal
service for one year from date of shipment. If the product proves defective during this period, C.B.S.
Scientific will repair or replace it at our option, free of charge, if returned to us postage prepaid. This
warranty does not cover: damage in transit, damage caused by carelessness, misuse or neglect, normal
wear through frequent use, damage caused by solvent corrosion, damage caused by improper handling
or user alteration, nor unsatisfactory performance as a result of conditions beyond our control. C.B.S.
Scientific shall in no event be liable for incidental nor consequential damages, including without limitation,
lost profits, loss of income, loss of business opportunities, loss of use and other related damages,
however caused, nor any damage arising from the incorrect use of the product.
ENGLISH
www.cbsscientific.com 4 Semi-Dry Blotter Manual 12/12
FRANÇAIS INFORMATION IMPORTANTE À L'USAGE DES UTILISATEURS
Le présent manuel d'utilisation explique la manière de se servir efficacement du produit en conditions
de sécurité. Il est recommandé de soigneusement lire la totalité du manuel, avec ses consignes
et ses instructions.
Le triangle avec point d'exclamation est un symbole destiné à avertir l'utilisateur du produit
de l'importance de certaines exigences relatives au fonctionnement, à l'entretien et/ou à la
garantie.
Le triangle avec flèche en zigzag est un symbole destiné à avertir l'utilisateur du produit de la
possibilité d'exposition à des décharges avec danger de secousses électriques.
Tout manquement à l'observation des consignes et des instructions peut exposer les personnes
et les biens à des dommages corporels et/ou matériels et peut ann uler to ute ga rantie. Il faut toujours
interrompre l'alim entation de coura nt continu avant de déconnecter les cordons d'alimen tation du
produit. Déconnecter d'abord les cordons d'alimentation branchés sur la source de tension
(alimentation de secteur) puis ceux branchés sur le produit. P our une sécurité maximum, il faut
toujours faire fonctionner ce système dans un lieu isolé, peu fré quenté, où le pe rsonnel non autorisé
n'a pas accès. Ne jamais faire fonctionner un matériel endommagé ou affecté par des fuites.
GARANTIE ET RESPONSABILITÉ
Le produit a été fabriqué conformément aux normes applicables les plus exigentes en matière de
matériaux, de main d'oeuvre, de conception et d'ingéniérie. C.B.S. Scientific garantit que le produit a
subi des essais et que ses performances rempliront les conditions des spécifications publiées ou leur
seront même supérieures. La présente garantie n'est valide que si le produit a fonctionné et a été
entretenu conformément aux consignes et instructions fournies.
C.B.S. Scientific g arantit qu e le produit sera dépourvu de vices de matériaux et de main d'oeu vre, en
conditions de service normales, pendant un an à compter de la date d'expédition. Au ca s où le produit
s'avérerait défectueux pendant cette période de garantie, C.B.S. Scientific réparera ou rem placera le
produit, à sa discrétion et gratuitement, si le produit lui est retourné port payé d'avance. La garantie ne
couvre pas les dommages de transport; les dommages causés par l'imprudence, le manque de soins,
l'abus ou la négligence; l'usure normale résultant d'une utilisation fréquente; les dommages causés par
la cor rosion des s olvants; et les dom mages c ausés par la ma nipulation inadéquate ou des
changements apportés par l'utilisateur. La garantie ne couvre pas non plus les performances non
satisfaisantes résultant de conditions hors du contrôle de C.B.S. Scientific. C.B.S. Scientific ne pourra
en aucun cas être tenue responsable de dommages indirects, y compris, de manière non limitative, la
perte de bénéfices, le manque à gagner, la perte d'occasions d'affaires, l'impossibilité d'usage ou tous
autres dommages associés, quelle qu'en soit la cause, ni de dommages résultant de l'usage incorrect
du produit.
ESPAÑOL INFORMACIÓN IMPORTANTE PARA EL USUARIO
El presente instructivo explica la manera de usar este producto en forma segura y efectiva. Sírvase
leerlo en su totalid ad y seguir detenidamente la s indicaciones q ue contiene.
El símbolo del triángulo con exclamación llama la atención del usuario a requisitos
importantes para el uso y mantenimiento del producto, así c omo p ara la validez de la
garantía.
El símbolo del triángulo con rayo llama la atención del usuario a la posibilidad de
riesgos eléctricos.
El incumplimiento de las instrucciones aquí señaladas podría dar lugar a riesgos a la persona, al
laboratorio o a ambos y podría an ular toda garantía. Siempre apague la fuente de corriente continua
antes de desenchufar los cables eléctricos del producto. Primero desconecte los cables de la fuente
de energía y después del producto. Para mayor seguridad, siempre use este sistema en un área
aislada, de poco movimiento de personas e inaccesible a personal no autorizado. Jamás use equipo
que presenta algún daño o fuga.
GARANTÍA Y RESPONSABILIDAD
Este producto fue fabricado de acuerdo con las normas más estrictas que sean factibles en cuanto a
materiales, mano de obra y diseño. C.B.S Scientific garantiza que se sometió el producto a pruebas y
que cumplirá o excederá las especificaciones publicadas. Esta garantía será válida únicamente si se
usa y se da servicio de mantenimiento al producto de acuerdo con las instrucciones señaladas.
C.B.S. Scientific garantiza que este producto se encontrará libre de defectos de materiales y mano de
obra por un período de servicio normal de un año a partir de la fecha de embarque. Si el producto
resulta defectuoso durante este período, C.B.S. Scientific lo re parará o lo repondrá, a criterio de
C.B.S., libre de cargos, si se devuelve el producto a C.B.S. porte pagado. Esta garantía no cubre
daños sufridos en tránsito, daños provocados por descuido, mal uso o negligencia, desgaste normal
como con secuencia del uso excesivo, daños atribuibles a corrosión provocada por solventes, daños
causados por el uso indebido o alteraciones realizadas por el usuario n i rendimiento insatisfactorio
atribuible a circunstancias fuera del control de C.B.S. Scientific. C.B.S. Scientific en ningún caso
asumirá responsabilidad por daños incidentales o subsecuentes, incluyendo, en forma no limitativa, la
pérdida de utilidades, de ingresos, de oportunidades comerciales o del uso del producto y otros daños
afines, fuere cual fuere su origen, ni por daños derivados del uso incorrecto del producto.
DEUTSCH WICHTIGE INFORMATION FÜR DEN BENUTZER
Diese Bedienungsanleitung beschreibt wie man dieses Produkt sicher und wirksam benutzt. Bitte
lesen und befo lgen Sie alle An weisungen in d ieser Anleitung .
Das Dreieck mit Ausrufezeichen weist den Benutzer des Produktes darauf hin, daß
wichtige Bedienungs-, Wartungs- und/oder Garan tievorschriften zu be achten sind.
Das Dreieck mit Zickzackblitz warnt den Benutzer des Produktes vor möglichen
Gefahren durch elektrische Spannungen.
Nichtbeachtung dieser Anweisungen kann zu persönlichen und/oder labortechnischen Schäden
führen und g leichzeitig alle Ga rantien als nich tig erklä ren. Die DC Stromzufuhr muß immer, vor dem
Entfernen der Stromkabel vom Produkt, abgeschaltet werden. D ie Stromzufuhrkabel müssen zuerst
von der Steckdose und erst dann vom Produkt entfernt werden. Um höchste Sicherheit zu
gewährleisten sollte dieses System in einem abgesonderten und besonders ruhigen Bereich eingesetzt
werden und vor Unbefugten sicher sein.
GARANTIE UND HAFTUNG
Dieses Produkt wurde unter Anwendung von Produkten mit höchster Qualität und aus Materialien mit
bester Verarbeitung und modernstem Design hergestellt. C.B.S Scientific garantiert, daß das Produkt
getestet w urde und alle publizierten Spezifikationen übertrifft. Diese Garantie ist jedoch nur gültig,
wenn das Produkt nach der beigefügten Bedienungsanleitung bedient und gewartet wurde.
C.B.S. Scientific garantiert, daß dieses Produkt bei normaler Bedienung aus fehlerfreiem Material
besteht und fehlerfrei in der Ausführung ist. Diese Garantie gilt für ein Jahr ab Liefe rdatum. S ollte das
Produkt in diesem Zeiraum fehlerhaft werden, bietet C.B.S. Scientific eine kostenlose Reparatur bzw.
kostenlosen Ersatz, einschließlich freiem Rückporto. Diese G arantie schließt folgendes aus:
Transportschaden, Schaden durch Nachlässigkeit, Mißbrauch oder Vernachlässigung, normale
Abnützung durch regelmäßigen Gebrauch, Schaden durch Säureangriff, Schaden durch falsche
Handhabung, Veränderung des Produktes durch den Benutzer, oder unzureichende Leistungen die
sich nicht im Verantwortungsbereich von C.B.S. Scientific befinden. C.B.S. Scientific kommt unter
keinen Umständen für folgende S chäden auf: Sachschadensverlust, E inkommensverlust, Verlust von
Geschäftsmöglichkeiten, Verlust der Anwendung und andere damit verbundene Schäden die auf
irgend eine Art un d Weise entstanden sind, o der Schäden die aus falscher Anwen dung des Produkte s
entstanden sind.
ITALIANO INF ORMAZIONI IM PORTANTI PER L’UTENTE
Questo manuale spiega come utilizzare questo prodotto in maniera sicura ed efficiente. Si pregai di
leggere e segu ire con cautela le istruzioni di ogni parte di questo m anuale.
Il triang olo co ntenete il simbolo di un pun to es clamativo avverte l’utente di importanti
requisiti relativi al funzionamento, man utenzione e/o ga ranzia del prodotto.
Il triangolo conten ete il sim bolo di u n lamp o avve rte l’uten te del pro dotto d ella
possibilità di pericoli dovuti a corrente elettrica.
La mancata osservanza delle istruzioni può essere causa di pericolo alla propria persona ed al
laboratorio, oltre a p oter annullare la g aranzia. Prima di distaccare il cordone d’alimentazione dal
prodotto, spegnere sempre la sorgente di corrente continua. Distaccare i cordoni d’alimentazione
prima dal lato della sorgente di tensione e poi dal lato del prodotto. Per maggior sicurezza, mettere
sempre in funzione il prodotto in un’area isolata con poco traffico che non sia accessibile al personale
non autorizzato. Non mettere mai in funzione un’apparecchiatura che sia danneggiata o abbia
perdite.
GARANZIA E RESPONSABILITÀ
Questo prodotto è stato fabbricato seguendo gli standard più elevati per i materiali, la manodopera e la
progettazione. La C.B.S. Scientific garantisce il prodotto è stato sottoposto a prova e raggiunge o
supera i valori pubblicati per i da ti tecnici. Questa garanzia è valida solo se il prodotto è messo in
esercizio e soggetto a manutenzione secondo le istruzioni fornite.
La C.B.S. Scientific garantisce che questo prodotto è libero di difetti di materiali e manodopera, in
normali condizioni d’esercizio, per la durata di un anno dalla data di spedizione. Se, in questo periodo,
il p rodotto si dimostre rà difettoso, la C.B.S. S cientific, a suo giudizio, lo r iparerà o sostituirà. Questa
garanzia non copre danni in transito, danni causati da negligenza, uso improprio, trascuratezza,
normale consumo derivante da uso frequente, o danni causati da solventi corrosivi, danni causati da
maltrattamento o da modifiche apportate dall’utente e non copre prestazioni insoddisfacenti che siano il
risultato di condizio ni al di fuo ri del controllo del fabbricante. La C.B.S. Scientific non s arà in ogni caso
responsabile per da nni incidentali o cons equenziali, incluso, senz a limitazioni, perdite di p rofitto,
perdita di entrate, perdita di opportunità d’affa ri e altri danni relativi, comunque causati, e per danni
risultati da uso incorretto del prodotto.
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SECTION 1
General Information
1.1 Introduction
The C.B.S. Scientific Semi-Dry Blotting Systems will transfer Western, Northern, or Southern
blots reliably and quickly. These blotting systems have transfer capabilities for gel dimensions up
to 12cm x 34cm (Cat. # EBU-3000), 20cm x 20cm (Cat. # EBU-4000) or 35cm x 45cm (Cat. #
EBU-6000). The stainless steel cathode and platinum-coated titanium anode are durable and
corrosion-resistant. These electrodes create a uniform electric field producing excellent transfers
onto nitrocellulose, supported nitrocellulose, nylon, charged nylon or activated paper substrates.
The upper electrode housing is separate from the base, allowing it to be secured at the desired
height with side hand screws. This unique design permits several gels to be transferred
simultaneously.
In the Semi-Dry system, blotting is accomplished by layering transfer membrane and gel between
buffer saturated blotting paper, minimizing the amount of buffer required. To prevent drawing
current away from the gel during a transfer, the uncovered portion of the cathode is shielded with
a Mylar® mask. This shield insures that all applied current passes directly through the gel. Use
of the mask will increase transfer efficiency and reduce transfer times up to 50%.
1.2 Specifications
Constructions:
Buffer chamber, safety cover Acrylic
Electrode panels Stainless steel, platinum-coated titanium
Electrode support panel Styrene
Power cords FR Urethane rated 7500VDC @ 200mA,
65°C
Mylar masks Mylar
EBU-3000
EBU-4000
EBU-6000
Shipping
weight 7lbs 7lbs
15lbs
Overall Size
(l)x(w)x(h) cm
15 ¾ x 7 ¾ x 2 ¾
9 ¾ x 9 ¾ x 2 ¾
19 ¾ x 17 x 2 ½
Maximum Gel Size - cm
12cm x 34cm
20cm x 20cm
35cm x 45cm
Distance between
electrodes -cm
1cm 1cm
1cm
Recommended Power
Supply
EPS-600
EPS-600
EPS-600
1.3 Safety
Power to the Semi-Dry Blotter is to be supplied by an external DC voltage power supply that must
be ground isolated so that the DC voltage output floats with respect to ground. For any power
supply used, the maximum specified operating parameters for the units are:
Maximum Limits
EBU-3000
EBU-4000 EBU-6000
50 VDC 50 VDC 50 VDC
500mA current 500mA current 1200mA current
55° C ambient temperature 55°C ambient temperature 55°C ambient temperature
Current to the unit, provided from the external power supply, must enter the unit through the lid
assembly, providing a safety interlock to the user. Current to the unit is broken when the lid is
removed. Do not attempt to use the unit without the safety lid, and always turn the power
supply off before removing the lid, or when working with the unit in any way. Follow
safety precautions specified by the power supply manufacturer.
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Section 2
Description of Parts
2.1 Unpacking the EBU-3000, EBU-4000 or EBU-6000
Please verify that your unit comes complete with the following components:
• Blotting chamber with stainless steel cathode, and platinum-coated anode
• Safety cover with power leads
• Mylar sheets (for shielding uncovered portion of anode)
2.2 Components/Assembly
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SECTION 3
Instructions for Use --- The instructions are divided into the following three categories:
A. General Considerations
B. Semi-Dry Electro-Blotting of DS DNA from Agarose Gels (Southern)
C. Semi-Dry Blotting of Proteins (Western)
D. Semi-Dry Blotting of RNA (Northern)
3.1 Blotting Unit Preparation
Place the blotting chamber on a level work surface in an authorized work area.
A. General Considerations
• Because transfer efficiency depends on many factors (e.g. gel concentration and
thickness, size, shape and net charge of molecule) results may vary. For resolving
gels, the % acrylamide range from 8 to 10% will separate with close to 100%
efficiency the transfer of 14 – 66 kD.
• Polyacrylamide gels have pore sizes that are too small for effective capillary diffusion
of DNA. These types of gels must be blotted electrophoretically requiring a transfer
buffer of low ionic strength (0.5 to 1X TBE). This is why nylon membranes are
generally used (uncharged nylon being preferable to positively charged nylon).
Nylon is the membrane of choice because it will bind DNA fragments as small as
50bp and can be irradiated to covalently linking the DNA to the membrane.
• The general “rule of thumb” for electrophoretic transfer in semi-dry blotting units is
application of a maximum current of 0.8mA/cm
2
of gel area. For gels used in the
EBU-3000 (10 x 30cm up to a max. of 12 x 34cm) or the EBU-4000 (15 x 15cm up to
a max. of 20 x 20cm) this translates to upwards of 300mA. In the larger EBU-6000,
this can increase to 1200mA. Please ensure that an adequate power supply is being
used to power the transfer.
• In general, most runs should take between 15 to 60 minutes depending on the size
and type of the gel and they type of transfer, i.e., Southern, northern or western.
• After setting the current at mA range, one can usually switch to the voltage panel to
obtain the voltage being applied. Most power supplies allow you to switch back and
forth between the amperage and voltage.
• Percent of acrylamide Size range transferred
(resolving gel)
(approx. 100% efficiency)
5-7 29-150 kD
8-10 14-66 kD
13-15 <36 kD
18-20 <20 kD
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B. Semi-Dry Electro-Blotting of DS DNA from Agarose Gels (Southern)
1.
Preparation for Southern Blotting
a. Agarose gels up to 6mm thick may be run in these systems. Thicker gels
require longer transfer times. Prior to transfer, gels should be stained
with Ethidium Bromide. Ethidium bromide is a Mutagen. ALWAYS wear
gloves. Dispose of solutions in accordance with the safety regulations of
your institution.
b. Optionally, depurinate the gel by soaking in 0.25M HCl for 20 minutes.
Depurination can increase transfer efficiency. To denature the DNA in
the gel before transfer, soak in 0.5M NaOH, 1.0M NaCl for 20 minutes.
Neutralize 2X for 15 minutes in 0.5M Tris-HCl, pH 7.4, 1.5M NaCl.
c. Membrane selection: Nylon or nylon supported nitrocellulose. ALWAYS
use forceps to handle membranes. If nylon is not base resistant,
denature DNA prior to transfer. If it is, you may base wash the DNA after
transfer instead of denaturing before. Base wash for 30 min. in 0.1N
NaOH followed by suspension in 0.2M Tris-base, pH 8.0, 0.5% SDS for
30 minutes to neutralize the membrane.
2. Preparation of the Transfer Materials
a. Required Materials: Membrane, cut to exact size of gel; saturate in ddH
2
O
for ten minutes, then in 0.1X TAE or 0.3X TBE for 20 minutes.
b. Gels should be soaked in running buffer, 0.1XTAE or 0.3XTBE for 20-60
minutes depending on thickness (3-6mm).
c. Cut eight sheets of 3mm blotter paper (Whatman) the exact size of the
gel. Saturate all eight sheets in running buffer.
d. Mylar Mask: Prepare a mask by cutting a rectangular hole that is slightly
smaller (1-2mm) than the gel dimensions.
e. For transfer of ssDNA (acrylamide): use 0.5 –1X TBE buffer. Equilibrate
in transfer buffer for 15 minutes.
3. Stacking of components into Electro-Blotter Apparatus:
a. Insert the mylar mask over the bottom platinum coated titanium electrode
(anode +). Note: The bottom electrode is the anode and is fixed in place,
while the cathode is adjustable and within the lid of the apparatus. Place
3-4 pieces of saturated filter paper carefully centered over the cut-out.
Flatten the filter paper by rolling a glass rod over the surface.
b. Apply a saturated membrane on the top of the paper.
c. Carefully place the gel on the membrane, making sure no bubbles remain
between the gel/membrane interface.
d. Stack the remaining 3-4 sheets of blotting paper on the top of the gel.
Add one sheet at a time and be sure to ‘smooth’ with glass rod to remove
air bubbles.
e. Lift the cathode assembly using the white handle in the center of lid; place
lid directly over the stack and apply downward pressure while tightening
each of the nylon thumb screws.
4. Power Setting
Connect the unit to the power supply using the power cords supplied. Warning:
C.B.S. Scientific has designed this device such that it cannot be opened unless
the power cords are disconnected. DO NOT attempt to open the transfer
apparatus without disconnecting the power cords and shutting off the power
supply.
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a. Transfer DNA of agarose gels (Southern) requires some special precautions.
Excessive heat build-up can melt agarose if too much power is applied. To
avoid this, try to use higher percentage gels made from high melt agarose.
b. With this type of agarose, run at low current (100-200mA for 15-30 minutes,
depending on the gel thickness.
c. If you are using low melt agarose or low percentage agarose, use 50mA for
45-60 minutes.
5. Single stranded DNA from 6% Acrylamide
a. 100-125mA @ constant current for 45-60 minutes (0.5-1XTBE)
B. Semi-Dry Blotting of Proteins (Western)
1. Preparation for Western Blotting
a. Acrylamide gels up to 3mm thick may be transferred in these systems.
b. Buffer System
192mM Glycine Towbin reference
25mM Tris, pH 8.3
0.0013M SDS
10-20% MeOH
39mM Glycine Maniatis reference
48mM Tris, pH 8.3
0.0375% (w/v) SDS
20% (v/v) Methanol
c. If the gel is to be stained with Coomasie Blue prior to blotting, refer to Perides,
et. al. (1986) for an alternate protocol before blotting.
2. Prior to completion of SDS-PAA electrophoresis:
a. Prepare a Mylar™ mask by cutting a rectangular hole that is slightly smaller
(1-2mm) than the gel dimensions.
b. Prepare 8 pieces of Whatman 3mm chromatography paper the exact size of
the gel. Larger pieces of filter paper may promote a ‘short circuit’ by allowing
current to bypass the gel and therefore reduce the efficiency of the transfer.
Soak the filter paper in transfer buffer until saturated. The ‘ready for use’
thickness should be around 1mm (dry paper is approximately .35mm).
c. Hydrate the Nitrocellulose by first floating in a tray of de-ionized water
(capillary absorption) followed by 5 minutes of total immersion.
3. Stacking of components into Electro-Blotter Apparatus
a. After SDS-PAA electrophoresis of proteins, separate the gel plate sandwich
and briefly soak the gel in a tray of de-ionized water. Remove the stacking
gel with a spatula or razor blade and discard.
b. Wet the anode and cathode plates by wiping with a lint free paper towel. No
puddles should be left in the unit.
c. Insert the Mylar mask over the bottom platinum coated titanium electrode
(anode +). Note: The bottom electrode is the anode and is fixed in place,
while the cathode is adjustable and within the lid of the apparatus. Place 3-4
pieces of saturated filter paper carefully centered over the cutout. Flatten the
filter paper by rolling a glass rod over the surface.
www.cbsscientific.com 10 Semi-Dry Blotter Manual 12/12
d. Apply a saturated membrane on the top of the paper.
e. Carefully place the gel on the membrane, making sure no bubbles remain
between the gel/membrane interface.
f. Stack the remaining 3-4 sheets of blotting paper on the top of the gel. Add
one sheet at a time and be sure to ‘smooth’ with glass rod to remove air
bubbles.
g. Lift the cathode assembly using the white handle in the center of lid; place lid
directly over the stack and apply downward pressure while tightening each of
the nylon thumb screws.
4. Power Settings
a. Calculate power input by using 0.8mA/sq. cm of gel for larger gel transfers.
Mini-gels (10 x 11cm) may be transferred at up to 100mA (5-25V) total
current. Remember, smaller proteins (<20kd) require less time than medium
size (<80kd) or larger proteins.
b. At the conclusion of the run, turn power supply off and disconnect leads from
blotter. Cover will not release unless power cord is removed. Remove the
cover slowly to catch any part of the gel sandwich sticking to the cathode.
After each use, clean the electrodes of buffer salts by rinsing in distilled
water.
C. Semi-Dry Blotting of RNA (Northern)
1. Electro-Blotting RNA
a. Buffer System:
1X TAE (40mM Tris-acetate, 1mM EDTA, pH8.0)
4.84g Tris-base
1.14ml glacial acetic acid
0.37g Na2EDTA-2H
2
O
pH to 8.0 with HOAC
or
1XTBE: (89mM Boric Acid, 2.5mM EDTA, pH 8.4)
10.8g Tris-base
5.5g Boric Acid
0.93g Na2EDTA-H
2
O
H
2
O to 1 liter
2. Preparation of the Gel
a. Formaldehyde must be removed from the gel by soaking in 0.1XTAE for at
least an hour at room temperature.
b. Standard electrophoresis buffers for RNA are usually 1XTAE or 1XTBE.
Electro-blotting transfer occurs in low ionic strength buffer. Exchange the 1X
electrophoresis bufferby soaking in 0.1X buffer for 20-30 minutes.
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3. Preparation of the Apparatus
a. Prepare 10 sheets of blotting paper cut to the same size of the gel and soak in
0.1TXAE. Cut a nylon membrane to the same size and wet in ddH
2
O for 5
minutes followed by equilibrate in 0.1XTAE for 20-30 minutes.
b. Prepare one of the clear plastic Mylar sheets (provided in the kit) by cutting a
hole in the sheet which is 1-2mm smaller than the gel dimension.
c. Insert the Mylar mask over the bottom electrode anode. Place 5 pieces of
saturated filter paper carefully centered over the cut-out in the Mylar mask. If
prepared correctly, the filter paper will overlap the mask cut-out slightly.
Flatten the filter paper by rolling a glass rod over the surface. Apply the
equilibrated Nylon membrane to the stack of filter paper followed by the gel
and 5 more pieces of saturated filter paper. Again roll a glass rod over the
filter paper stack to compress and remove trapped air bubbles. Lift the
cathode assembly using the white handle in the center of lid; place lid directly
over the stack and apply downward pressure while tightening each of the
nylon thumb screws.
4. Electro-blotting transfer
a. Once the lid is in place, insert the power cords into the apparatus. Plug the
ends of the power cords into the Power Supply.
b. Set the Power Supply at 250mA and transfer for 30 minutes at constant
current.
c. After the run, turn off the power supply and disconnect the power cords from
the apparatus. The lid cannot be lifted unless the power cord is unplugged
from the anode. Carefully lift the lid and watch for filter paper sticking to the
top electrode. Remove the Nylon membrane and process according to
protocol. Rinse the inside of the apparatus and the mask with ddH
2
0.
www.cbsscientific.com 12 Semi-Dry Blotter Manual 12/12
SECTION 4
References
1. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith,
J.A.,Struhl, K. (ed) (1993). Current Protocols in Molecular Biology. Vol. 2, Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc., Ch.10.
2. Bjerrum, O.J., Schafer-Nielsen, C., Buffer systems and transfer parameters for semi-
dry electroblotting with a horizontal apparatus, Electrophoresis ’86, 315-327, VCH,
Weinheim, Germany.
3. Dunn, S.D., Effects of the modification of transfer buffer composition and the
renaturation of proteins in gels on the recognition of proteins on Western blots by
monoclonal antibodies, Anal. Biochem., vol. 157, 144-153, (1986).
4. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning. A Laboratory
Manual. 2
nd
edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York. 18.47-18.61.
5. Towbin, J., Staehelin, T., Gordon, J., Electrophoresis transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: Procedure and some applications,
PNAS, USA, Vol. 76, 4350-4354 (1979).
www.cbsscientific.com 13 Semi-Dry Blotter Manual 12/12
SECTION 5
Maintenance of Equipment
5.1 Care and Handling
The plastic components of the Semi-Dry Blotting units are fabricated from acrylic and polycarbonate.
Electrodes and connectors are made from pure platinized titanium, and stainless steel. As with any
laboratory instrument, adequate care ensures consistent and reliable performance.
After each use, rinse with de-ionized water. Wipe dry with a soft cloth or paper towel, or allow to air
dry. Whenever necessary, all components may be washed gently with water and a non-abrasive
detergent, and rinsed and dried as above. Never use abrasive cleaners, glass cleaning sprays or
scouring pads to clean the components, as these will damage the unit and components.
Additional precautions:
• Do not autoclave or dry-heat sterilize the apparatus or components.
• Do not expose the apparatus or components to phenol, acetone, benzene,
halogenated hydrocarbon solvents or alcohols.
• Avoid prolonged exposure of the apparatus or components to UV light.
• Do NOT treat with diethylpyrocarbonate (DEPC)-treated water for extended
periods at 37°C.
• Electrostatic charge and heat affix the conductive platinum surface covering the
titanium. It can be scratched off or damaged by using sharp objects or scouring
pads. This will disturb the continuity of the electric field and have a negative
effect on transfers.
• The stainless electrode can be permanently damaged by corrosion if the polarity
between anode and cathode is reversed. CAUTION: Be certain of power
connections before initiating transfer.
5.2 Maintenance
The following inspection and maintenance procedures will help maintain the safety and reliable
performance of the Blotting systems. Replacement parts can be ordered by calling 1-858-7554959 or by contacting your local distributor.
• Banana plugs and power cords should be inspected regularly. If the banana
plugs become loose or do not feel friction tight replace the plugs or power cords.
• Should power cord assemblies (connectors, wire or shrouds) show any signs of
wear or damage (e.g. cracks, nicks, abrasions, or melted insulation), replace
them immediately.
• The platinum wire is secured to the banana jack by compression between a
stainless washer and the jack nut. The nut/washer interface should be tight and
free of corrosion.
www.cbsscientific.com 14 Semi-Dry Blotter Manual 12/12
SECTION 6
Equipment and Accessories
Cat. # Item
EBU-3000 Semi-Dry Blotting System. Includes power leads and safety interlock, 12cm x 34cm.
EBU-4000 Semi-Dry Blotting System. Includes power leads and safety interlock, 20cm x 20cm.
EBU-6000 Semi-Dry Blotting System. Includes power leads and safety interlock, 35cm x 45cm.
EPS-600 Power Supply, 96-240 VAC, 500-60 Hz, 5-600V, 3-500mA
MM-4000 Mylar® Mask for EBU-4000, package of 4 each
MM-6000 Mylar® Mask for EBU-6000, package of 6 each
NOTES
CONTACT INFORMATION
Telephone:
Local or International
858-755-4959
Toll Free: 800-243-4959
Fax: 858-755-0733
Online ordering:
www.cbsscientific.com
E-mail address:
sales@cbssci.com
Mailing address:
C.B.S. Scientific Company
P.O. Box 856
Del Mar, CA 92014
Shipping address:
10805 Vista Sorrento Parkway
Ste 100
San Diego, CA 92121
Credit Card Options:
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Discover/American Express
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