C.B.S. Scientific DSG User Manual

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INSTRUCTION MANUAL

Dual Vertical Slab Gel Systems

DSG-125-02 DSG-160-02 DSG-170-02 DSG-190-02 DSG-200-02 DSG-250-02 DSG-400-02

TABLE OF CONTENTS

Page

Important User Information 3-4 Section 1 General Information

1.1 Introduction 5

1.2 Specifications 5

1.3 Safety 6

Section 2 Description of parts

2.1 Unpacking 7

2.2 Components/Assembly 7

Section 3 Instructions for Use

3.1 Dual Slab Gel Unit Preparation 8

3.2 Preparation/Cleaning of glass plates 8

3.3 Gel Casting 8-10

3.4 Running the Gel 10-11

3.5 Removing the Gel 11

3.6 Maximum Well/Comb volumes 11

Section 4 Running Conditions

4.1 Recommended Power 12

4.2 Recommended Buffer & Reagents 12-13

4.3 References 13

Section 5 Maintenance of Equipment

5.1 Care and Handling 14

5.2 Maintenance 14 Section 6 Ordering Information 15-18

C. B. S.  Scientific 2 Dual Slab Gels

ENGLISH

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

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.

electrical exposure.

C. B. S.  Scientific 3 Dual Slab Gels

FRANÇAIS INFORMATION IMPORTANTE À L'USAGE DES UTILISATEURS

Le présent manuel d'utilisation explique la manière de se ser vir efficacement du produit en c onditions

de sécurité. Il est recommandé de soigneusem ent 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 instruc tions peut exposer les personnes et les biens à des dommages corporels et/ou m atériels et peut annuler toute garantie. Il faut toujours interrompre l'alimentation de courant continu avant de déconnecter les cordons d'alimentation du produit. Déconnecter d'abord les cordons d'alimentation branchés sur la source de tension (alimentation de secteur) puis ceux branchés sur le produit. Pour une sécurité m aximum, il faut toujours faire fonctionner ce système dans un lieu isolé, peu fréquenté, où le personnel non autorisé n'a pas accès. Ne jamais faire fonctionner un matériel endommagé ou affecté par des fuites.

GARANTIE ET RESPONSABILITÉ

Le produit a été fabriqué conformément aux norm es 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 r empliront les conditions des spéc ifications publiées ou leur seront même supérieures. La présente garantie n'est valide que si le produit a fonctionné et a été entretenu conformément aux consignes et instructions fournies.

C.B.S. Scientific garantit que le produit sera dépourvu de vices de matériaux et de main d'oeuvre, en conditions de service normales, pendant un an à com pter de la date d'expédition. Au cas où le pr oduit s'avérerait défectueux pendant cette période de garantie, C.B.S. Scientific réparera ou r emplacera 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'im prudence, le manque de soins, l'abus ou la négligence; l'usure normale résultant d'une utilisation fréquente; les domm ages causés par la corrosion des solvants; et les dommages causés par la manipulation inadéquate ou des changements apportés par l'utilisateur. La garantie ne couvre pas non plus les perf ormances non satisfaisantes résultant de conditions hors du contrôle de C.B.S. Scientific. C.B.S. Scientific ne pourr a en aucun cas être tenue responsable de dommages indirects, y compris, de m anière non limitative, la perte de bénéfices, le manque à gagner, la perte d'occasions d'aff aires, l'impos sibilité d'usage ou tous autres dommages associés, quelle qu'en soit la cause, ni de dom mages résultant de l'usage incorrect du produit.

ESPAÑOL INFORMACIÓN IMPORTANTE PARA EL USUARIO

El presente instructivo explica la manera de usar este producto en f orma segura y efectiva. Sírvase leerlo en su totalidad y seguir detenidamente las indicaciones que contiene.

El símbolo del triángulo con exclamación llama la atenc ión del usuario a requisitos importantes para el uso y mantenimiento del producto, así como para la validez de la garantía.

El símbolo del triángulo con rayo llama la atención del usuario a la posibilidad de riesgos eléctricos.

El incumplimiento de las instrucciones aquí señaladas podría dar lugar a riesgos a la persona, al laboratorio o a ambos y podría anular toda garantía. Siempre apague la fuente de corriente continua antes de desenchufar los cables eléctric os del producto. Primero desc onecte los cables de la fuente de energía y después del producto. Para mayor seguridad, siempre use este sistem a 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 especif icaciones publicadas. Esta garantía s erá 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 m ateriales y mano de obra por un período de servicio normal de un año a partir de la fecha de embar que. Si el producto resulta defectuoso durante este período, C.B.S. Scientific lo reparará o lo repondrá, a criterio de C.B.S., libre de cargos, si se devuelve el producto a C.B.S. porte pagado. Esta garantía no cubre daños sufridos en tránsito, daños provocados por descuido, mal uso o negligencia, desgaste norm al como consecuencia del uso excesivo, daños atribuibles a corrosión provocada por solventes, daños causados por el uso indebido o alteraciones realizadas por el usuario ni rendimiento insatisfac torio 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, inc luyendo, en forma no limitativa, la pérdida de utilidades, de ingresos, de oportunidades comerciales o del uso del pr oducto y otros daños afines, fuere cual fuere su origen, ni por daños derivados del uso incorrecto del producto.

DEUTSCH WICHTIGE INFORMATION FÜR DEN BENUTZER

Diese Bedienungsanleitung beschreibt wie man dieses Produkt sicher und wirksam benutzt. Bitte lesen und befolgen Sie alle Anweisungen in dieser Anleitung.

Das Dreieck mit Ausrufezeichen weist den Benutzer des Produktes darauf hin, daß wichtige Bedienungs-, Wartungs- und/oder Garantievorschriften zu beachten sind.

Das Dreieck mit Zickzack blitz warnt den Benutzer des Produktes vor möglichen Gefahren durch elektrische Spannungen.

Nichtbeachtung dieser Anweisungen kann zu persönlichen und/oder labortechnisc hen Schäden führen und gleichzeitig alle Garantien als nichtig erklären. Die DC Stromzufuhr muß imm er, vor dem Entfernen der Stromkabel vom Produkt, abges chaltet werden. Die Stromzufuhrkabel müssen zuerst von der Steckdose und erst dann vom Produkt entfernt werden. Um höchste Sicherheit zu gewährleisten sollte dieses System in einem abgesonderten und besonders ruhigen Ber eich eingesetzt werden und vor Unbefugten sicher sein.

GARANTIE UND HAFTUNG

Dieses Produkt wurde unter Anwendung von Produkten mit höchster Qualität und aus Materialien m it bester Verarbeitung und modernstem Des ign hergestellt. C.B.S Scientific gar antiert, daß das Produk t getestet wurde und alle publizierten Spezifikationen übertrifft. Diese Garantie ist jedoch nur gültig, wenn das Produkt nach der beigefügten Bedienungsanleitung bedient und gewartet wurde.

C.B.S. Scientific garantiert, daß dieses Produkt bei normaler Bedienung aus fehlerfreiem Material besteht und fehlerfrei in der Ausführung ist. Diese Garantie gilt für ein Jahr ab Lieferdatum. Sollte das Produkt in diesem Zeiraum fehlerhaft werden, bietet C.B.S. Scientif ic eine kostenlose Reparatur bzw. kostenlosen Ersatz, einschließlich freiem Rückporto. Diese Garantie schließt folgendes aus: Transportschaden, Schaden durch Nachlässigkeit, Mißbrauch oder Vernachlässigung, normale Abnützung durch regelmäßigen Gebrauch, Schaden durch Säureangriff, Schaden durch falsche Handhabung, Veränderung des Produktes durch den Benutzer, oder unzureichende Leistungen die sich nicht im Verantwortungsbereich von C.B.S. Scientific befinden. C.B.S. Sc ientific kommt unter keinen Umständen für folgende Schäden auf: Sachschadensverlust, Einkommensverlust, Verlust von Geschäftsmöglichkeiten, Verlust der Anwendung und andere damit verbundene Schäden die auf irgend eine Art und Weise entstanden sind, oder Schäden die aus falscher Anwendung des Produktes entstanden sind.

ITALIANO INFORMAZIONI IMPORTANTI PER L’UTENTE

Questo manuale spiega come utilizzare questo prodotto in maniera sicura ed eff iciente. Si pregai di leggere e seguire con cautela le istruzioni di ogni parte di questo manuale.

Il triangolo contenete il simbolo di un punto esclamativo avverte l’utente di impor tanti requisiti relativi al funzionamento, manutenzione e/o garanzia del prodotto.

Il triangolo contenete il simbolo di un lampo avverte l’utente del prodotto della

laboratorio, oltre a poter annullare la garanzia. Prima di distaccare il cordone d’alimentazione dal prodotto, spegnere sempre la sorgente di corrente continua. Distacc are 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 s ia acces sibile 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 s tandar d più elevati per i materiali, la manodopera e la progettazione. La C.B.S. Scientific garantisce il prodotto è stato sottopos to a prova e raggiunge o supera i valori pubblicati per i dati tecnici. Questa garanzia è valida solo se il prodotto è messo in esercizio e soggetto a manutenzione secondo le istruzioni fornite.

La C.B.S. Scientific garantisce che questo prodotto è liber o di difetti di materiali e manodopera, in normali condizioni d’esercizio, per la durata di un anno dalla data di spedizione. Se, in questo periodo, il prodotto si dimostrerà difettoso, la C.B.S. Scientific, a suo giudizio, lo riparerà o sostituirà. Questa garanzia non copre danni in transito, danni causati da negligenza, uso improprio, trascuratezza, normale consumo derivante da uso frequente, o danni causati da s olventi corrosivi, danni causati da maltrattamento o da modifiche apportate dall’utente e non copre pres tazioni insoddisfacenti c he siano il risultato di condizioni al di fuori del controllo del fabbricante. La C.B.S. Scientific non sarà in ogni caso responsabile per danni incidentali o consequenziali, incluso, senza limitazioni, perdite di profitto, perdita di entrate, perdita di opportunità d’affari e altri danni relativi, comunque causati, e per danni risultati da uso incorretto del prodotto.

possibilità di pericoli dovuti a corrente elettrica.

La mancata osservanza delle istruzioni può essere causa di pericolo alla propria persona ed al

C. B. S.  Scientific 4 Dual Slab Gels

SECTION 1 General Information

1.1 Introduction

C.B.S. Scientific offers the Dual Slab Gel Systems designed to meet a wide variety of applications. These systems are designed for separating and characterizing protein and can be adapted for short sequencing runs with the appropriate accessories. Two gels can be run simultaneously under identical buffer conditions. The cooling chamber assures constant temperature during electrophoresis. Gel casting can be accomplished using our convenient and unique Gel Wrap™, or by using our multiple gel casting chambers (see Section 6, Dual Slab Gel Kits and Accessories).

With the use of special accessories, two dimensional (2-D) gel electrophoresis can be performed the 220mm vertical system (cat. # DSG-200-02). First dimension I.E.F. tube gels are run on the unit using IEF Tube Gel Adapters (described in the catalogue). Buffer surrounds the tubes and is cooled by the internal cooling chamber for temperature-controlled runs. Second dimension separation is performed by using the standard accessories. A complete description of 2-D protocol is included when ordering the I.E.F. Tube Gel Adapters (cat. # TGA-200K).

Other applications include; mobility shift assays, antibody super shift assays, Rnase protection assays, DNA footprinting, differential display, DNA paternity testing and forensic analysis.

1.2 Specifications Constructions:

Buffer chamber, safety cover Acrylic

Electrodes Platinum wire .012" diameter

Power cords FR Urethane rated 7500VDC,

200mA, 65°C Combs Teflon Glass plates Soda-lime float glass Spacers PVC Clamps Polypropylene, stainless steel Safety Certification EN61010-1-1993 (IEC1010-1)

Table 2 Specifications

Dual Slab Gel Unit:

145mm

160mm

170mm

190mm

220mm

280mm

387mm

Shipping weight

12 lb. 24 x 23 x 21.91 250 300 upper

12 lb. 24 x 23 x 23.33 265 300 upper

12 lb. 24 x 23 x 24.29 275 300 upper

12 lb. 24 x 23 x 26.35 295 300 upper

12 lb. 24 x 23 x 29.12 325 300 upper

13 lb. 24 x 23 x 35.24 385 300 upper

16 lb. 24 x 23 x 45.94 480 300 upper

Overall unit size cm (l) x (w) x (h)

Distance between electrodes mm

Recommended buffer volume mls

125-200 lower

Voltage Limit VDC

725

800

850

950

1100

1400

1900

C. B. S.  Scientific 5 Dual Slab Gels

1.3 Safety

Power to the Dual Slab Gel units 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

60 watts power 80mA current 55°C ambient temperature

power supply used, the maximum specified operating parameters for the units are:

Maximum Limits

50VDC/cm of gel

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 alw ays turn the power

supply off before removing the lid, or when working w ith the unit in any w ay. Follow safety precautions specified by the power supply manufacturer.

SECTION 2

C. B. S.  Scientific 6 Dual Slab Gels

Description of Parts

2.1 Unpacking

Please verify that your unit comes complete with the following components:

Dual Slab Gel Kit:

• Dual Slab Gel electrophoresis unit

• Safety cover with attached DC power leads

• 4 ea GPC-0001, white spring clamps

• 12 ea GPC-0002, white spring clamps

• 2 ea comb

• 2 set glass plates

• 2 set spacers

• 2 ea Gel Wrap

• Bubble level, attached to base

• 4 ea leveling screws, nylon

2.2 Components/Assembly

Inlet Coolant Port

Outlet Coolant Port

Safety Cover

Clamp

Electrophoresis Unit

4mm Power Cord Assembl

Comb

Glass Plate

Spacer

C. B. S.  Scientific 7 Dual Slab Gels

SECTION 3 Instructions for Use

3.1 Dual Slab Gel Unit Preparation

1. Place Dual Slab Gel unit on a level work surface in an authorized work area. Level the unit by adjusting the four white nylon thumbscrew until the bubble is centered in the level. Be sure that all four of the screws are touching the bench.

2. Verify that the comb, spacer set and Gel Wrap™ gasket are the same thickness, by assembling without gel casting.

3. To use the internal cooling option, attach tubing to the inlet (bottom port) and outlet coolant ports (top port) and to your pump.

3.2 Preparation/Cleaning of Glass Plates

Hand wash both plates with a high quality lab detergent followed by a complete rinsing with dH

O. Air dry or use a lint-free tissue. Spray/wipe the chosen inner surfaces of the plate set with

95% ethanol and dry with lint-free tissue.

3.3 Gel Casting Using Gel Wrap™ Gasket Casting method

1. Start by holding the rectangular back plate with the rounded bottom corners and start applying the gasket around one side of the glass plate. Note: one side of the “U” shaped gasket is flat, and the other side has tubing that will act as a seal around the spacers.

2. When applying the gasket over the rounded corners of the back glass plate, make sure the notches on the gasket align with the rounded corners of the glass plate. Once the gasket is pushed over the bottom edge and corners, work it down the remaining side.

3. Place the gasketed plate on the lab bench with the tubing side up, and extend the bottom of the plate over the edge of the bench, approximately ¾ of an inch. Place the spacers along side the inside edges of the gasket. Be sure the rounded corner end of each spacer is facing the outside bottom of the plate, following the radius of the glass.

C. B. S.  Scientific 8 Dual Slab Gels

4. Place the notched plate on top of the bottom assembly, starting from the bottom edge and gently easing the plate down. Verify the gasket is smooth around the edges and then clamp along the bottom.

5. Lift the assembly and stand it on the base of the clamps. For leveling, push glass plate assembly down until it stops against clamp body. Clamp the sides of the assembly with additional casting clamps on either side. As each clamp is attached, be sure the gasket is aligned between the plates forming a seal.

6. Apply PAGE or agarose solution to gel plate sandwich using a syringe or pipette. If using a stacking gel, pour desired height of running gel, then overlay a small amount of dH

O or 0.1% SDS solution to top of gel. After polymerization, rinse with buffer,

add stacking gel solution and insert comb. For regular, unit percentage gels, add polyacrylamide solution to correct height, and insert comb. Allow gel to set, usually 20 minutes. Extra gel solution in pipette or syringe can be monitored to test polymerization of gel mix.

7. Disassembly. Hold the clamped plate assembly with one hand. Remove the gasket by starting at one of the top ends and pulling up and out on the gasket until it releases from the plate, up to the bottom of each of the white clamps. When each clamp is reached DO NOT remove it, instead feed the gasket down through the clamp body and repeat pulling up and out. Continue feeding until the gasket is fully detached. If gel, is not to be used immediately, wrap entire plate sandwich with plastic wrap tightly to seal and store at 4°C for up to a month.

C. B. S.  Scientific 9 Dual Slab Gels

3.4 Running the Gel

1. Attach plate set(s) to unit with smaller or notched plate facing towards the upper buffer reservoir. If using a precast gel stored at 4°C, allow to warm to room temperature. Use white clamps with mouth open (cat. # GPC-

0001) to attach plate set to unit, clamping each side to the sandwich.

2. Pour freshly prepared buffer in upper and lower chamber. Using a pipette or syringe, thoroughly flush out the wells in the glass plate sandwich with buffer from the upper chamber. Boil or heat samples and then place immediately on ice. Load samples. If outer lanes do not contain sample, it is recommended that you run standards (Rainbow™ standards can be monitored easily during run) and/or fill outer lanes with loading buffer to reduce smiling and wraparound effects.

3. Attach safety cover.

4. Connect the leads to the power supply matching the color-coded red to red and black to black. See

Section 4.1 for recommended

power conditions.

Begin separation by electrophoresis.

C. B. S.  Scientific 10 Dual Slab Gels

3.5 Removing the Gel

1. Turn the power supply off and disconnect the leads from the power supply. Remove the safety cover from the unit, by placing thumbs on white posts next to red & black connectors, then pushing down while pulling up with fingers under lid. DO NOT pull on power cords.

2. Drain upper reservoir using a pipette or syringe. Detach side clamps and remove gel sandwich. Carefully separate, making sure that the gel remains attached to one plate. Stain and fix according to your preferred method. Dispose of buffer according to your institutions safety requirements.

3.6 Maximum Well/Comb Volumes

NOTE: To calculate sample well volume expressed in millimeters (mm) of height

divide maximum volume by tooth depth.

TABLE 1---Combs

Tooth

Overall Length

13cm

1 130.2 19.1 3.2 1865 2486 3729 4966 7458 2 63.5 19.1 3.2 909 1213 1819 2426 3639

3 equal 41.3 19.1 3.2 591 788 1182 1576 2364

2 w/middle

marker

4 30.1 19.1 3.2 432 576 1151 1152 1728 5 23.5 19.1 3.2 336 449 673 898 1347 6 19.1 19.1 3.2 273 364 546 728 1092

8 13.5 19.1 3.2 193 257 386 514 771 10 10.1 19.1 3.2 145 194 291 388 582 12 7.9 19.1 3.2 113 151 227 302 453 14 6.4 19.1 3.2 91 121 182 242 363 15 5.7 19.1 3.2 82 109 163 218 327 16 5.2 19.1 3.2 74 98 147 196 294 20 4.2 17.7 2.4 55.72 74.3 111.45 148.7 222.9 29 2.9 17.7 1.6 38.5 51.33 77 102.66 154.0 30 2.8 17.7 1.6 37.2 49.6 74.4 99.12 148.8

Tooth width/

59.8/

4.2

Spacing

depth

between teeth mm

19.1 3.2 856 1142 1713 2284 3426

0.75mm

thickness

volume per

tooth

microliters

(ul)

1.0mm

thickness

volume per

tooth

microliters

(ul)

1.5mm

thickness

volume per

tooth

microliters

(ul)

2.0mm

thickness

volume per

tooth

microliters

(ul)

volume per

3.0mm

thickness

tooth

microliters

(ul)

C. B. S.  Scientific 11 Dual Slab Gels

SECTION 4 Running Conditions

4.1 Recommended Power

Precise electrophoresis conditions will vary according to the number and type of gels used, buffer conditions employed, power input, and the general goal of the experiment. Refer to the reference section for in depth discussions on practical and theoretical approaches to protein gel electrophoresis.

Using standard SDS-PAGE buffer systems apply 1-10 VDC/cm of gel. For sequencing applications use 50 VDC/cm. If running two gels in the Dual Units, keep the volts the same but double the mA. It is also true that if the thickness of gel increases, increase the mA proportionally.

At constant voltage, the proteins will migrate at a constant rate during electrophoresis with adequate heating appropriate for denaturing gels. Increasing the voltage/mA (for a single gel thickness and percentage) will speed mobility but increase the risk of overheating.

The sample migration rate can be increased by raising the input power. This can be done son systems which employ “active” temperature control such as Dual Slab Gel Units and Dual MiniVertical Gel Units. The joule heating generated by the higher input power is offset by the cooling effect of the water jacket between the gels. Exact conditions should be determined empirically but could be increased at least in the 20% range.

4.2 Recommended Buffers and Reagents

Pre-mixed acrylamide stock solutions are the method of choice. Use according to manufacturer’s instructions.

Typical ‘scratch’ recipe for a 4% acrylamide gel:

10mls 40% acrylamide

6.6mls 2% Bis-acrylamide stock

5mls 10X TBE

78.4mls dH

750ul 10% APS 50ul TEMED

1. Make up 0.5X TBE buffer

2. After gel apparatus is set-up and ready for the gel to be poured, add 750ul fresh 10% APS solution to the acrylamide solution.

3. Add 5 to 10 ul TEMED and using a 10 to 25ml pipette, quickly “pour” the gel.

4. Allow the gel to polymerize at least 60 minutes.

5. Remove the comb after polymerization and wash out wells with 0.5X TBE (acrylamide will seep into the wells).

6. Fill upper and lower chambers with 0.5X TBE.

7. Pre-electrophorese gel, if needed, 20-30 minutes.

8. Load wells with samples.

9. Monitor migration with dye markers.

C. B. S.  Scientific 12 Dual Slab Gels

4.2 Recommended Buffers and Reagents-continued

BUFFERS: Agarose Page/Agarose Slab

TBE (1X solution):

TAE (1X):

0.089M Tris base 0.04M Tris-acetate

0.89M Boric acid 0.001M EDTA

0.002M EDTA pH 8.0 pH 8.3

Protein Denaturing Denaturing/Non-Denaturing/

TG-SDS(1X):

Ny lon Blotting

0.025M Tris base TT (Tris-Taurine( (1X): TT-SDS (1X):

0.192M Glycine 0.1M Tris base 0.1M Tris base

0.1%(w/v) SDS 0.1M Tricine 0.1M Tricine pH3 0.1%(w/v) SDS

DNA Sequencing

TTE (Tris/Taurine/EDTA)(1X):

1.78M Tris

0.57M Taurine

0.01M EDTA Na2-2H

4.3 References

Hames, B.D., Rickwoood, D. (ed.) (1990). Gel Electrophoresis of Proteins. A Practical Approach.

Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. 2

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.,

edn. IRL Press, Oxford. Ch.1 & 3.

edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 18.47-18.61.

Struhl, K. (ed)

(1993). Current Protocols in Molecular Biology. Vol. 2, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., Ch.10.

C. B. S.  Scientific 13 Dual Slab Gels

SECTION 5 Maintenance of Equipment

5.1 Care and Handling

Additional precautions:

The plastic components of the Dual Slab Gel units are fabricated from acrylic and polycarbonate. Electrodes and connectors are made from pure platinum, stainless steel, and chrome plated brass. As with any laboratory instrument, adequate care ensures consistent and reliable performance.

After each use, rinse buffer chamber, glass plates, spacers and combs 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.

• 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. A brief rinse with DEPC-water is sufficient after a thorough wash.

5.2 Maintenance

The following inspection and maintenance procedures will help maintain the safety and reliable performance of the Dual Slab Gel systems. Replacement parts can be ordered by calling 1-858-4959 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.

C. B. S.  Scientific 14 Dual Slab Gels

SECTION 6 Dual Slab Gel Kits and Accessories

Cat. # I Item DSG-125 or DSG-125-02 Dual Slab Gel Kit, 145mm height. Includes 2 combs, 2 Gel Wrap spacer sets, 2 Gel