C.B.S. Scientific DGGEK-2401 User Manual

Download

Cipher DGGE Electrophoresis Systems

DGGEK-1001 DGGEK-2001 DGGEK-2401

INSTRUCTION MANUAL

TABLE OF CONTENTS

Important User Information . . . . . . . . . . . . . . . . . . . . . . . .3-4

Section 1: General Information

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.2 Specications

1.3 Safety

Section 2: DGGEK Unit Set-Up

2.1 Unpacking Instructions

2.2 Filling DGGEK Tank with Buffer

2.3 Heater/Stirrer/By-Pass Pump (Controller) Instructions

2.4 Setting Buffer Temperature

2.5 Connecting Power Supply

Section 3: Gel Casting Options

3.1 Preparation of Glass Plates . . . . . . . . . . . . . . . . . . . . . . 16

3.2 Gel Casting Techniques . . . . . . . . . . . . . . . . . . . . . .16-20

3.3 Vertical Gradient Formation

3.4 Perpendicular Gel Casting

Section 4: Gel Running Preparations

4.1 Preparation of Cassettes

4.2 Buffer Cycling Set-Up . . . . . . . . . . . . . . . . . . . . . . 25-26

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

. . . . . . . . . . . . . . . . . . . . . . . . . . . 5

. . . . . . . . . . . . . . . . . . . . . . .6-7

. . . . . . . . . . . . . . . . . . . . 7

. . . . . . . . . . 8-13

. . . . . . . . . . . . . . . . . . . . . . 14

. . . . . . . . . . . . . . . . . . . . . 14-15

. . . . . . . . . . . . . . . . . . . 21

. . . . . . . . . . . . . . . . . . . 22-23

. . . . . . . . . . . . . . . . . . . . . . 24

Section 5: Running Conditions

5.1 Running the Gels

5.2 Removing the Gels . . . . . . . . . . . . . . . . . . . . . . . 27

5.3 Buffer Disposal . . . . . . . . . . . . . . . . . . . . . . . . . 27

Section 6: Recommended Buffers

6.1 Stock Solutions

Section 7: Comb Specications . . . . . . . . . . . . . . . . . . . . . 29

Section 8: References

Section 9: Troubleshooting

Section 10: Maintenance of Equipment

10.1 Care and Handling . . . . . . . . . . . . . . . . . . . . . . . . 32

10.2 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Section 11: Ordering Information . . . . . . . . . . . . . . . . . . . . 33

Section 12: Regulatory Compliance

. . . . . . . . . . . . . . . . . . . . . . . . . 26

. . . . . . . . . . . . . . . . . . . . . . . . . 28

. . . . . . . . . . . . . . . . . . . . . . . . . 30

. . . . . . . . . . . . . . . . . . . . . . . 31

. . . . . . . . . . . . . . . . . . . . 34-35

Cipher DGGE Instructions 2/28/14

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 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

rst and then from the product. For maximum safety, always operate this system in an isolated, low trafc area, not accessible to unauthorized personnel. Never operate dam-

aged or leaking equipment.

WARRANTY AND LIABILITY

This product was produced utilizing the highest practical standards of materials, work-

manship, and design. C.B.S. Scientic warrants that the product has been tested and will meet or exceed published specications. This warranty is valid only if the product

has been operated and maintained according to the instructions provided.

C.B.S. Scientic 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. Scientic 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 con-

ditions beyond our control. C.B.S. Scientic shall in no event be liable for incidental nor consequential damages, including without limitation, lost prots, 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.

www.cbsscientic.com

FRANÇAIS INFORMATION IMPORTANTE À L’USAGE DES UTILISATEURS

Le présent manuel d’utilisation explique la manière de se servir efcacement 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 èche 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 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 maximum, 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 normes applicables les plus exigentes en matière de matériaux, de main d’oeuvre, de conception et d’ingéniérie. C.B.S. Scientic garantit que le produit a subi des essais et que ses performances rempliront les conditions des spécications 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. Scientic garantit que le produit sera dépourvu de vices de matériaux et de main d’oeuvre, en conditions de service normales, pendant un an à compter de la date d’expédition. Au cas où le produit s’avérerait défectueux pendant cette période de garantie, C.B.S. Scientic réparera ou remplacera 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 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 performances non satisfaisantes résultant de conditions hors du contrôle de C.B.S. Scientic. C.B.S. Scientic ne pourra en aucun cas être tenue responsable de dommages indirects, y compris, de manière non limitative, la perte de bénéces, 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 totalidad y seguir detenidamente las indicaciones que contiene.

El símbolo del triángulo con exclamación llama la atención 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 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 Scientic garantiza que se sometió el producto a pruebas y que cumplirá o excederá las especicaciones 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. Scientic 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. Scientic 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 normal 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 insatisfactorio atribuible a circunstancias fuera del control de C.B.S. Scientic. C.B.S. Scientic 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 anes, 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 Zickzackblitz warnt den Benutzer des Produktes vor möglichen Gefahren durch elektrische Spannungen.

Nichtbeachtung dieser Anweisungen kann zu persönlichen und/oder labortechnischen Schäden führen und gleichzeitig alle Garantien als nichtig erklären. Die DC Stromzufuhr muß immer, vor dem Entfernen der Stromkabel vom Produkt, abgeschaltet 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 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 Scientic garantiert, daß das Produkt getestet wurde und alle publizierten Spezikationen übertrifft. Diese Garantie ist jedoch nur gültig, wenn das Produkt nach der beigefügten Bedienungsanleitung bedient und gewartet wurde.

C.B.S. Scientic 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. Scientic 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. Scientic benden. C.B.S. Scientic 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 efciente. 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 importanti requisiti relativi al funzionamento, manutenzione e/o garanzia del prodotto.

Il triangolo contenete il simbolo di un lampo avverte l’utente del prodotto della 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 poter annullare la garanzia. 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 trafco 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. Scientic garantisce il prodotto è stato sottoposto 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. Scientic 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 prodotto si dimostrerà difettoso, la C.B.S. Scientic, 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 solventi corrosivi, danni causati da maltrattamento o da modiche apportate dall’utente e non copre prestazioni insoddisfacenti che siano il risultato di condizioni al di fuori del controllo del fabbricante. La C.B.S. Scientic non sarà in ogni caso responsabile per danni incidentali o consequenziali, incluso, senza limitazioni, perdite di protto, perdita di entrate, perdita di opportunità d’affari e altri danni relativi, comunque causati, e per danni risultati da uso incorretto del

prodotto.

Cipher DGGE Instructions 2/28/14

SECTION 1 General Information

1.1 Introduction

Denaturing Gradient Gel Electrophoresis (DGGE) is a powerful genetic analysis technique that can be used for detecting single base changes and polymorphisms in genomic (1, 2), cloned, and PCR amplied DNA (3,4). Two of the most valuable uses for DGGE in human genetics are in directly detecting single base changes that cause disease and in detecting polymorphisms with DNA probes for genetic-linkage analysis. Also, DNA fragment melting points can be determined using Perpendicular DGGE (1).

C.B.S. Scientic has designed four different Cipher DGGEK Systems, which are reliable and easy-to-use. The DGGEK-1001 is a 2 gel system and features one dual gel cassette. The DGGEK-2001 features two single gel cassettes. The DGGE-2401 has the same buffer tank as the DGGE-1001 and DGGE-2001 but features two dual gel cassettes, for a capacity of 4 gels per run. Finally there is the DGGE-4801, which features the larger buffer tank and four dual cassettes enabling the researcher to run up to 8 gels simultaneously. Improvements to the technique include: a simplied method for casting perpendicular and vertical gels using Gel Wrap™. Single or dual gel cassettes have been redesigned eliminating the use of agarose plugs, the need for an external peristaltic pump for buffer cycling has been eliminated, bulky screw clamps have been replaced with polypropylene spring clamps, buffer tank dimensions have been changed to use bench space more efciently, and a safety cover with an electrical

interlock has been added for protection to help maintain temperature and reduce evaporation.

1.2 Specications

Constructions

DGGE Tank Acrylic DGGE Lid Sodalime glass, acrylic

Electrodes Platinum wire .010” diameter Power cords FR Silicone rated 7500V, 200mA, 65° C DGGE Cassette Acrylic Heater/Stirrer Buffer Cycler Stainless steel

Combs Teon Spacers UHMW-PE Glass Plates Sodalime or Borosilicate

Gradient Maker Acrylic, teon, stainless steel

ADDITIONAL SPECIFICATIONS

Model Cat. #

# of cassettes

Max. # of gels

gel dimensions 15cm(w) x 20cm(l) 15cm(w) x 20cm(l) 15cm(w) x 20cm(l)

Optimal Buffer

Volume

Minimum

Buffer Volume

Required

footprint (cm)

Cipher DGGEK-1001 Cipher DGGEK-2001 Cipher DGGEK-2401

1 dual cassette 2 single cassettes 2 dual cassettes

2 gels 2 gels 4 gels

21 Liters -1 dual cassette 21.5 Liters - 1 single cassette

21 Liters - 2 single cassettes

18 Liters 18 Liters 18 Liters

25.4(w) x 54.6(l) x 34.3(h) 25.4(w) x 54.6(l) x 34.3(h) 25.4(w) x 54.6(l) x 34.3(h)

21 Liters - 1 dual cassette 20 Liters - 2 dual cassettes

www.cbsscientic.com

1.3 Safety

Power to the DGGE systems is to be supplied by an external DC voltage power supply

that must be ground isolated so that the DC voltage output oats with respect to ground. For any power supply used, the maximum specied operating parameters for

the units are: Maximum Limits

250 VDC voltage 30 watts power 80 mA current 70°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. DC current to the unit is broken when the lid is opened. Do not attempt to use the unit without the safety

lid. Always turn the power supply off before removing the lid, or when working with the unit in any way. Follow safety precautions specied by the power supply

manufacturer.

Input Power: Mains to safety interlock

Depending on country of destination, input voltage from mains electrical supply are as follows: 110-120VAC @ 50/60Hz/5 Amps or 200-240VAC @ 50/60Hz/5 Amps. Country

specic power cords or CE approved adapter kits are supplied with each system.

Warning: Do NOT turn on Heater/Stirrer until tank has been lled with buffer!

SECTION 2 DGGEK Unit Set-Up

2.1 Unpacking Instructions

1) Unpack the DGGE Tank Assembly and place on level surface in an approved location. Using gure 2-1 verify that your DGGEK-1001 or DGGEK-2001 System comes complete with the following components:

• EPS-300 X Power Supply (not pictured)

• Peristaltic Mini-Pump for gel casting

• Lower Reservoir/Acrylic tank/safety interlock/ with 2 black leads inside lid

• Heater/Stirrer/By-pass pump (Controller) - factory-mounted onto DGGE tank

• Gel cassette(s), combs, spacer sets, Gel Wrap™ gaskets, and glass plates sets as specied in chart below:

Type of Gel Cassette(s)

DGGEK-1001 1 - dual 2 2 2 2 4 2

DGGEK-2001 2- single 2 2 2 2 4 2

DGGEK-2401 2 - dual 4 4 4 4 8 4

• 1 Gradient maker, 20mls per side, Cat. # GM-40

• Buffer displacement tank (DGGEK-1001 only, not pictured)

• White spring clamps

received with system will vary depending on format of DGGE)

• Buffer siphon pump with tubing, Cat# BSP-1000

• 1 Conversion Plate (not shown) for running 1 gel at a time on dual cassette (for DGGEK-1001, DGGEK-2401 only)

# of Sample Well Combs

# of 1-well Combs

# of Gel Wrap ™ Spacer Sets

# of Perpendicular Spacer Sets

Cipher DGGE Instructions 2/28/14

# Gel Wrap™ Gaskets

# glass Plate Sets

Figure 2-1

Heater Stirrer

Buffer Siphon Pump Assembly

Vapor Shield

Thermometer

DGGE Tank

Single Cassetes

Comb (2)

Spacers (2 pairs)

Glass Plate Assembly(2)

Clamps

Mini-Pump (Optional)

GM-40

Gelwrap

(2)



Buffer recycling manifold

2. Open the lid and conrm the following: a) Two lengths of small bore silicone tubing for buffer cycling are already attached

to the buffer recycling manifold outlets

b) A single length of large bore tubing from the By-Pass Pump is connected to the

2.2 Filling DGGE tank with Buffer

barb-tting on the plastic manifold.

DGGEK-2001

1) Fill the tank with running buffer (see Section 6 for buffer formulations) to optimal

level for number and type of cassettes used. Refer to chart below for exact buffer volumes required: This requires 20 to 21.2 liters of buffer. See chart below:

Model Cat. #

Optimal Buffer

Volume

Cipher DGGEK-1001 Cipher DGGEK-2001 Cipher DGGEK-2401

21 Liters -1 dual cassette 21.5 Liters - 1 single cassette

Use distilled or RO water when making buffer. NOTE: Buffer Displacement Tank Instructions:

With DGGE-1001 - ll buffer displacement tank, BDT-1000 with water and place in

With DGGE-2001 or DGGE-2401- If using only 1 cassette, BDT-1000 can also be

the back of DGGE tank.

used to save buffer but must be ordered separately.

21 Liters - 2 single cassettes

21 Liters - 1 dual cassette 20 Liters - 2 dual cassettes

www.cbsscientic.com



 

2.3 Operating Heater/Stirrer/By-Pass Pump (Controller)

1) Attach power cord to heater/stirrer and plug into wall outlet.

(comes factory-mounted onto DGGE tank)

2.3.1 Front and Rear Panels

Front View Rear View

1. Display 11. Pump Speed Switch

2. Preset Temperature Buttons 12. Identification Label

3. Safety Set Reset Button 13. Pump Inlet

4. Safety Set Indicator Knob 14. Reservoir Purge

5. Cooling Light

15. Pump Ou tlet 2

6. Power ON / OFF Button 16. AC Input

7. Heating Light 17. Circuit Breaker / AC Power Switch

8. °F Light

9. Select/Set Knob (Push to set, turn to adjust)

10. °C Light

Present on R efrigera ting/Hea ting mode ls only

Built in on reservoir models only

Cipher DGGE Instructions 2/28/14

 

1. Temperature Sensor

2. Pump Shaft and Impeller

3. Pump Outlet Nozzle

4. Over-Tem perat ure Sensor

5. Heater Coils

Rotates on Immers ion Circulators

 

2.3.2 Controller Assembly

1. Temperature Sensor

2. Pump Shaft and Impeller

3. Buffer By-Pass Tubing Adapter

4. Over-Temperature Sensor

5. Heater Coils

2.3.3 Controller Specications

Temperature Stabil ity ±0.05°C

Readout Accuracy ±0.5°C

Over-Tem peratur e Protection Yes, user- adjustabl e

Low-Liqu id Protection Yes

Heater 1100W – 115V, 1600 W – 240V

Pump Speeds 2-speed, 9 liters per minute or 15 liters p er minut e

Environmental Conditions:

 Indoor Use Only  Over Voltage: Category ll  Maximum Altitude: 2000 meters  Operating Ambient: 5° to 30°C  Relative Humidity: 80% for temperatures to 30°C  Pollution Degree: 2  Class 1: Residential, Commercial , Light Industrial  Class 2: Heavy Industrial



www.cbsscientic.com

2.3.4 Controller Operation

2.3.4.a Reservoir Liquid Level

Fill the reservoir with the appropriate buffer. The DGGEK Systems are designed to be used with reagent buffers such as 1xTAE or 1xTBE. On Circulating Baths, the liquid level should

be sufcient to cover the heating coils, pump, over-temperature sensor, and at least one inch

(25mm) of the temperature sensor. On Immersion Circulators, the liquid level should be suf-

cient to fully immerse the heater coils, over-temperature sensor, and pump outlet nozzle.

2.3.4.b Power

An IEC power cord is provided with the Circulator. This power cord should be plugged into the IEC receptacle on the rear of the Controller and then plugged into a properly grounded outlet. Make sure that the power outlet is the same voltage and frequency indicated on the identication label on the back of the Controller. The use of an extension cord is not recommended. However, if one is necessary, it must be properly grounded and capable of handling the total wattage of the unit. The extension cord must not cause more than a 10% drop in voltage to the Circulator. Once the unit has been connected to an appropriate electrical

outlet, place the Circuit Breaker/Power Switch on the rear of the Controller in the ON position. Four decimal points (….) will appear on the digital display. DO NOT place the Power Switch on the front of the Controller ON until the Safety Set has been adjusted to the desired tem-

perature (see 3.6 below).

Indicated Voltage:

Volts/Phase/Frequency Operational Voltage Range Phase Frequency

100/ 1 / 60 90 to 110 Volts single 60 Hz

100/ 1 / 50 90 to 110 Volts single 50 Hz

120/ 1 / 60 110 to 130 Volts single 60 Hz

230/ 1 / 60 208 to 230 Volts single 60 Hz

240/ 1 / 50 220 to 240 Volts single 50 Hz

2.3.4.c Setting the Safety Set Set Point - NOTE: DGGEK is factory set to 80ºC

The Safety Set feature automatically disconnects Controller power to the heater and pump in the event that the reservoir liquid level drops too low or the Controller fails. The Safety Set is

user-adjustable between approximately 40° and 210°C. It should be set at least 5°C higher than the Software High Limit temperature. Use a at blade screwdriver to rotate the Safety

Set Indicator Knob to the desired temperature. Do not force the knob beyond the stops at either end of the temperature value range. Once the Safety Set temperature has been set, turn

power to the Controller ON by pressing the Power Switch on the front of the Controller. The pump will begin operating, the display will ash the current temperature set point (tx.xx), the

°C LED will light, and the current bath temperature will appear on this display. Pump speed selection is made using the Pump Speed Selection Switch on the rear of the Controller (see Section 2.3 or 3.3). If power is disrupted because the Safety Set temperature was exceeded, place the Circuit Breaker/Power Switch in the OFF position, press the Safety Set Reset Button, correct the problem (low liquid level, incorrect Safety Set temperature, etc.), and then restore power. Activation of the Safety Set during normal operation will display a fault (FLt 3)

on the readout.

2.3.4.d Selecting Temperature Units

The control set point and actual bath temperatures may be displayed in either °C or °F. The factory-default is °C. To change from °C to °F, place the Circuit Breaker/Power Switch on the rear of the Controller in the OFF position and then press and hold the P2 Button while turning

the power back ON.

Cipher DGGE Instructions 2/28/14

To change from °F to °C, place the Circuit Breaker/Power Switch in the OFF position and then

press and hold the P3 Button while turning the power back ON.

NOTE: When the temperature display units are changed, the Software High Limit value and all

temperature presets revert to the factory-default values. If a calibration value has been entered, the value will be retained.

2.3.4.e Setting the Software High Limit - NOTE: DGGEK is factory set to 75ºC

This feature provides additional safety and protection by allowing a selectable upper temperature limit set point. To avoid an unwanted shutdown during regular operation, the high limit value should be set at least 5°C higher than the selected control temperature To set the Software High Limit temperature set point, press the P2 and P3 keys simultaneously and repeat until (Hxxx) appears on the display. This is the current Software High Limit value. It is factory set at 75°C. To change the displayed value, press and turn the Select/Set Knob until the desired Software High Limit set point value is displayed. A clockwise rotation increases the value; a counterclockwise rotation decreases the value. Press the Select/Set Knob a second time to accept the new value and return to normal operation. If the Software High Limit value meets or

exceeds the control temperature set point, (E-H1) will ash on the display. If this occurs, enter

a higher value for the Software High Limit or reduce the control temperature set point. If the actual bath temperature reaches the Software High Limit setpoint, (FLt1) will ash on the display. Should this occur, the Controller will automatically remove power from the heater and, in Refrigerating/Heating units, the compressor as well. The pump will continue to operate. Once the problem is corrected (bath temperature reduced or Software High Limit value increased), press the Power button to clear the message.

2.3.4.f Setting the Set Point Temperature

Press and release the Select/Set Knob. The decimal point ashes to indicate that the set point

temperature can be changed. Turn the Select/Set Knob until the desired temperature set point is displayed. A clockwise rotation increases the setting; a counterclockwise rotation decreases the setting. Press the Select/Set Knob a second time to accept the displayed value. The deci-

mal point stops ashing and the display will indicate the actual bath temperature. Allow sufcient time for the bath to stabilize at the desired temperature before making any adjustments to set point temperature. NOTE: The unit will automatically accept the displayed set point after

approximately 10 seconds of inactivity, even if the Select/Set Knob was not pressed. The set point temperature may be checked at any time by pressing the Select/Set Knob. If the set point temperature cannot be raised, it is possible that the Software High Limit value is set lower than the desired control temperature set point. Reset the Software High Limit value to 5°C or more above the desired set point temperature.

2.3.4.g User-Denied Preset Temperatures

With the unit on, press the desired Preset Button — P1, P2, or P3. The LED associated with the selected

Preset Button will begin to ash. Rotate the Select/

Set Knob to the desired temperature set point. Press the selected Preset Button a second time to enter the new set point. The new set point temperature will not be saved unless the Preset Button is pressed. The LED associated with Preset Button lights continuously whenever that preset value is controlling bath temperature. If more than one Preset Button is set at a given temperature set point, the LED associated with all Preset Buttons with that set point will light.

www.cbsscientic.com

2.3.4.h Local Lockout Feature

 

This feature enables the user to lock all controls on the controller. While the feature is activated, the unit will remain running at the current settings. To activate the local lockout feature, press and hold the Select/Set Knob for 10 seconds. Once locked, the screen will read LLo.

When locked, the setpoint decimal point will not ash as usual. Press and hold the Select/Set

Knob again for 10 seconds to unlock the controls. Once unlocked, the screen will read CAn.

2.3.4.i Stick-on Strips for Preset Buttons

The Standard Controller is supplied with two stick-on strips that can be applied beneath the Preset Buttons. These strips are removable and can be reapplied. The user can write the set point temperature associated with each Preset Button on this strip. The use of an erasable

medium, such as dry erase marker or air tip

pen is recommended. The former can be wiped off with a dry cloth or tissue; the later can usually be removed with simple glass cleaner and a cloth or tissue. Do not use a permanent marker or ballpoint pen. Always do an ink test before writing on these strips.

The stick-on strip ts beneath the controller’s Preset Buttons. It is removable and reapplicable.

2.3.5 Controller Display Messages

Display Description Action Required

….

tx.xx Power up s elf-test Normal — Appears momentarily at startup

oCx.x Calibration offset v alue Normal — C urrent c alibration offset value; refer to section 6.1

Hxxx Software High Limit value Normal — C urrent S oftware High Limit value; refer to section 5.6

Axxx Auto-Refrigeration set point value

E-H1 Softwar e High Limit set point too low

FLt 1 Software High Limit valu e exceeded

FLt 2 EEPROM reset

FLt 3 Safety Set temperatur e exceeded

FLt 4 H eating Triac f ailure Error — Servic e required

FLt 5 Pr obe failure Error — Service required

FLt 6

Standby mode

i2c error — Commun ication f ailure to modulati on board

Normal — Indic ates that the Circuit Breaker/P ower Switc h is ON and the Controller Power Switch is OFF

Normal — Curr ent Auto-Refrigeration set point; refer to section

5.10 (Appears only on Refrigerating/H eating Circulators)

Error — The value entered is below the c ontrol temperature set point. Refer to Setting Software High Limit, s ection 5.6

Error — Set a Software High Limit value higher, then turn main power to the unit OFF and back ON; refer to section 5.6

Error — Turn Circuit Breaker/Power Switch O FF, hold P3 B utton, and then turn th e Circuit Break er/Power Switch back ON

Error — Check fluid level Check flui d temper ature and s et point Ensure th at OTP s et point is higher than fluid set point or increase to maximu m Turn Circuit Breaker/Power Switch OFF, press S afety Set R eset Button, and then turn the Circuit Breaker/P ower Switch back ON

Error — Service required (App ears onl y on Refrig erating/H eating Circulators)

Cipher DGGE Instructions 2/28/14

2.3.6 Controller Calibrations and Maintenance

2.3.6.a Calibration Calibration allows the user to match the Controller’s bath temperature display to an external reference thermometer. Calibration is performed as follows: Set the desired operating uid temperature set point and allow temperature to stabilize. Press the P2 and P3 simultaneously and release and repeat until the display reads (oCx.x). Press P1 and hold until (Cal) is displayed. This will take about 2 seconds. At one second intervals, the displayed value will alternate between

the actual bath uid temperature and the current offset value, which is the difference between

the factory calibration setting and the user’s reference temperature sensor. The maximum offset is ±0.9°C from factory calibration. To change the calibration offset value, rotate the Select/Set Knob until the display matches the reading on the reference temperature sensor. The display will continue to alternate between the offset value and the calibrated display temperature. Press the Select/Set Knob or the P1 Button to accept the entered value. When the new calibration is

stored and the mode is exited, (dONE) will appear on the display. NOTE: The displayed offset

value will also be accepted if there no activity for 20 seconds. 6.2 Heater The heater should be kept clean. If deposits build up on the heater, they may be removed by scrubbing with a nonmetallic (plastic) abrasive pad. Do not use steel wool.

2.3.6.b Pump Motor The pump bearings are permanently lubricated with high-temperature silicone grease and do not require additional lubrication. Should the bearings become noisy, replacement of the entire pump

motor is recommended. This will reduce repair labor costs and retain uid pumping reliability. A

replacement pump and motor mounting kit is available (see Section 10 - Replacement Parts). 6.4 Cleaning Only mild detergents and water or an approved cleaner should be used on the painted and stainless steel surfaces of the Circulator. Do not allow cleaning liquids or sprays to enter the Controller vents. A concentrated bath cleaner is available that can be used to remove mineral deposits from the reservoir. See Section 10 - Replacement Parts.

2.3.7 Controller Reservoir Fluids

For your buffers use distilled or RO water only!

DO NOT USE the following uids:

1. Automotive antifreeze with additives**

2. Hard tap water**

3. Deionized water with a specic resistance > 1 meg ohm

4. Any ammable uids

5. Concentrations of acids or bases

6. Solutions with halides: chlorides, uorides, bromides, iodides or sulfur

7. Bleach (Sodium Hypochlorite)

8. Solutions with chromates or chromium salts

9. Glycerine

10. Syltherm uids

** At temperatures above 40°C, additives or mineral deposits can adhere to the heater. If deposits are allowed to build up, the heater may overheat and fail. Higher temperatures and higher concentrations of additives will hasten deposit build up

Warning: Do not use a ammable liquid as a bath medium as a re hazard may result.

www.cbsscientic.com

DGGEK Unit Set-Up, con’t.

2.4 Setting Buffer Temperature

Warning: Do NOT turn on Heater/Stirrer until tank has been lled with buffer!

1. Turn Heater/Stirrer “On” and set temperature to 60°C according to

Section 2.3.4.f Setting the Set Point Temperature (page 11).

2.5 Connecting the Power Supply to Safety Interlock

2. Position EPS-300 X Power Supply on a shelf above the tank. Refer to Figure 2-2 to complete the steps below:

a. Attach the power supply to the safety interlock.

b. Attach the safety interlock to a wall (mains) outlet.

c. Do NOT attach power leads (from power supply to safety

interlock) until gels are loaded and installed in the tank.

Cipher DGGE Instructions 2/28/14

Power Supply Connection

To mains (wall outlet)

Figure 2-2

www.cbsscientic.com

SECTION 3 Gel Casting Options

3.1 Preparation/Cleaning of Glass Plate for Gel Casting

Hand wash both plates with a high quality lab detergent followed by a complete rinsing with dH2O. 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.

3.2 Gel Casting Techniques.

A. Gel Wrap™ Gasket Casting Method. B. Vertical gradient gel casting using GM-40 gradient maker and gravity ow. C. Vertical gradient gel casting using GM-40 gradient maker and a Mini-pump. D. Vertical gradient gel casting using GM-40 gradient maker, Mini-pump and

Multi-gel Caster.

NOTE: Prior to casting, mark the notched glass plate to designate which side is the ‘inside’ and ‘outside’. The same side of the notched plate should always sit against the reservoir gasket.

A. Gel Casting using Gel Wrap™ Gasket Casting method

For Vertical DGGE, use the set of spacers which do not have the small hole or channel milled into the lower end, these are for casting perpendicular gels.

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 at, 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.

Cipher DGGE Instructions 2/28/14

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.

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 theedges 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.

www.cbsscientic.com

3.2 Gel Casting Techniques- continued

C-1

C-2

V-1

V-2



B. Vertical gradient gel casting using GM-40 gradient maker and gravity ow.

1. Place the GM-40, gradient maker, on an elevated magnetic stirrer with a small “ea” stir bar in the cylinder (C-2) closest to the outlet. The gradient maker should be tted with a leur valve, (V-2) and a 20ga needle with attached tubing to deliver acrylamide to the gel plate sandwich as shown in gure (3.5). With the height differential between the leur valve (V-2) and the top of the gel plate sandwich between 2-4 inches, it takes approximately 5-9 minutes to cast the gel. Raising the height of the gradient maker can increase ow rate. Flow can be regulated by turning leur valve (V-2). Flow rate appropriate for each gradient should be determined empirically.

Fig. 3-5

Cipher DGGE Instructions 2/28/14

3.2 Gel Casting Techniques- continued

C. Vertical gradient gel casting using gradient maker (GM-40) and a mini-

pump.

1. Alternatively, you may choose to use a “mini-pump” or other peristaltic

pump to cast gels as shown in g (3.6). If so, secure the gradient maker to a ring stand and connect the outlet tubing to the mini-pump tubing adapter. Connect tubing from mini-pump to a 20ga. needle for afxing between glass plates.

Fig. 3-6

www.cbsscientic.com

3.2 Gel Casting Techniques- continued D. Vertical gradient gel casting using GM-40 gradient maker, Mini-Pump and

Multi-Gel Caster.

1. Clean the gel caster with soap and water and the glass plates with alcohol.

2. Place a separating sheet in the gel caster.

3. Place the back plate (non-eared) on the sheet.

4. Place the spacers on each side of the back plate.

5. Place the top plate (eared) on the spacers to form 1st sandwich. Repeat as required. Continue to Section 3.3 Vertical Gradient Formation.

Fig. 3-7

Cipher DGGE Instructions 2/28/14

3.3 Vertical Gradient Formation

To determine the range of gradient appropriate for your fragment analysis, please read the enclosed paper by Myers, Shefeld and Cox, especially section 6.1.3 through 6.2.1 (pages 124 to 126). This gives you an excellent overview of the determination of melting behavior of your fragments.

1. The following is a typical protocol for casting a 40%-60% gradient gel. Refer to Section 3.2 Vertical Gradient Gel Casting, gure 3.5, for apparatus assembly. In an ice bucket, place two 50ml conical tubes labeled “A” and “B”. Add to tube “A”

• 11.5ml of 40%/7.5%

• 80 µI (10%) APS

• 5 µl TEMED

2. Add to tube “B”:

• 11.5ml of 60%/7.5%

• 80 µI (10%) APS

• 5 µI TEMED

3. Pour solution "B" into right side of gradient maker (C-2),(GM-40) as shown in g. 3.5, and open interior valve to allow air bubble to escape. Let as much as 1 ml "B" solution BACKFLOW into left side of gradient maker. Decant the 1 ml back into right side with pasteur pipette. Remove any residual solution from (C-1) with absorbent paper.

4. Add solution "A" to left side of gradient maker.

5. Turn on magnetic stirrer.

6. Exit tube with needle should be secured to gel plate notch with tape.

7. Open inside valve rst (V-1), then outside (V-2) valve to start gravity ow. Optionally, Turn on mini-pump and pump acrylamide at medium speed. Determine optimal speed empirically.

8. Gel volume is 23ml, using the 0.75mm spacers. If gel volume is not enough to ll sandwich, use 0% to "top-off". If using water saturated butanol for overlay, leave 0.5cm void to create at interface.

9. Insert the sample well comb. Allow the gel to polymerize for 20-30 minutes.

Go to Section 4 for Gel Running Preparation.

www.cbsscientic.com

FIGURE 1

FIGURE 2

1.0mm AND THICKER

.75mm AND THINNER

PERPENDICULAR GEL CASTING

TO GRADIENT MAKER

20 GAUGE NEEDLE

SEAL WITH AGAROSE

HOLE THROUGH SPACER

GEL PLATE

CLAMPS

TO GRADIENT MAKER

20 GAUGE NEEDLE

SEAL WITH AGAROSE

GEL PLATE

CLAMPS

3.4 Perpendicular Gel Casting

NOTE: Prior to casting, mark the notched glass plate to designate which side is the

‘inside’ and ‘outside’. The same side of the notched plate should always sit against the reservoir gasket.

1. For perpendicular gel casting, locate the spacer which has a channel machined into it on one end which will be referred to as the “channel spacer”. The gel sandwich will be cast on its side, with the channel spacer on top and the channel further away from the single well comb (see gure 1 for casting gels 1.0mm and thicker, see gure 2 for casting gels .75mm or thinner).

2. Follow the Gel Wrap instructions 1-5 (pages 16-17), making sure to use the

channel spacer.

3. Insert single well comb into glass notch. A small amount of agarose is needed in two places once the gel sandwich is placed on its side, both places are located on the lower corner where the single well comb interfaces the spacer, and on the other side of the spacer where it interfaces the gel wrap.

4. Once the agarose has set, insert the gradient maker outow tube tipped with a 20ga. through the Gel Wrap and into the “channel” (gure 1), or space created between the horizontal spacer and the comb (gure 2). You may use the same piece of Gel Wrap many times (20-

30) for the perpendicular gels: do not use a piece of Gel Wrap Gasket for Vertical casting after you have poked a hole in it for perpendicular casting—it will leak.

Cipher DGGE Instructions 2/28/14

3.5 Perpendicular Gradient Formation

To determine the range of perpendicular gradient appropriate for your fragment

analysis, please read the enclosed paper by Myers, Shefeld and Cox, as well as the Methods and Enzymology V. 212 paper by Abrams and Stanton. This gives you an excellent overview of the determination of melting behavior of your fragments.

2. The following is a typical protocol for casting a 40%-60% gradient gel. Refer to Section 3.2 Vertical Gradient Gel Casting, gure 3.5 for apparatus assembly. In an ice bucket, place two 50ml conical tubes labeled “A” and “B”. Add to tube “A”

• 11.5ml of 40%/7.5%

• 80 µI (10%) APS

• 5 µl TEMED

2. Add to tube “B”:

• 11.5ml of 60%/7.5%

• 80 µI (10%) APS

• 5 µI TEMED

3. Pour solution "B" into right side of gradient maker,(GM-40), and open interior valve to allow air bubble to escape. Let as much as 1 ml "B" solution BACKFLOW into left side of gradient maker. Decant the 1ml back into right side with pasteur pipette. Remove any residual solution with absorbent paper.

4. Add solution “A” to left side of gradient maker.

5. Turn on magnetic stirrer.

6. Exit tube should be attached to near side of gel plate with tape.

7 Open inside (V-1) valve rst, then outside (V-2) valve to start ow.

8. Gel volume is 23ml, using the 0.75mm spacers. Allow 20-30 minutes for gel polymeraztion. If gel volume is not enough to ll gel sandwich, use 0% to “topoff”.

Go to Section 4 for Gel Running Preparation.

www.cbsscientic.com

SECTION 4 Gel Running Preparations

4.1 Preparation of the Cassettes

1. After polymerization rinse gel plate assembly with D.I. water to remove excess acrylamide or denaturants from plate exterior.

2. Remove comb and quickly transfer gel sandwich to cassette by removing all #2 clamps. LEAVE GEL WRAP IN PLACE. The Gel Wrap acts as a barrier and prevents perpendicular electrical elds from interfering with outside lanes. Reclamp to cassette using #1 clamps/4 per side. Dislodge bottom portion of Gel Wrap from the plates to allow buffer contact with the gel. See gure 4-1a. NOTE: If running only 1 gel on a dual cassette, attach conversion plate with clamps to second side of cassette.

Fig. 4-1a

3. Immerse single (g. 4-1b) or dual cassette(s) (g. 4-1c) into pre-heated buffer tank.

Fig. 4-1b

Fig. 4-1c

Cipher DGGE Instructions 2/28/14

4.2 Buffer Cycling Connections

1. Fill upper reservoir(s) by attaching recirculating tubing to barb tting on each upper reservoir (g. 4-2c,d,e). Rinse loading interface of gel with tank buffer to remove non-polymerized acrylamide or excess urea. Adjust nal level of lower reservoir buffer by lling the tank until the level of buffer reaches the underside of the upper reservoir.

Buffer Cycling Connections – Two Single Cassettes

Fig. 4-2c

Fig. 4-2d

www.cbsscientic.com

4.2 Buffer Cycling Connections-continued.

Buffer Cycling Connections – Dual Cassettes

Fig. 4-2e

SECTION 5 Running Conditions

5.1 Running the Gels

1. Load samples at 1:1 with neutral dye. Load 5-10 ug Genomic DNA/well or 1-2 ug cloned (B-globin)/well. Determine concentration by O.D. 260.

2. Attach black power leads to cassettes as shown in Fig. 5-1.

3. Close lid to engage safety interlock. Turn on power supply to 150V (40mA) constant Volts for 5-7 hours.

Fig. 5-1

Cipher DGGE Instructions 2/28/14

5.2 Removing the Gels

3. After the run is completed, turn off power supply, disconnect recirculating tubes and power leads, remove cassettes, remove glass sandwiches from cassettes.

4. Using a wedge plate separator,cat. # WPS-100, pry plates apart and immerse gel/ plate in buffer tray. Stain with Safe Stain or EtBr (.5ug/ml) for 5-10 minutes. Lift oating gel on plate out of tray and rinse with ddH20. Flip over onto saran wrap and peal off glass plate. View bands on transilluminator. Electro-Blot or photograph.

5.3 Recommended Power

1. 150V (40mA) CV for 5-7 hours

5.4 Disposal of Buffer Using Buffer Siphon Pump

NOTE: Buffer can be used several times before discarding. Determine empirically.

Dispose of solutions in accordance with the safety regulations of your institution.

1. Attach pump assembly to corner of tank as shown in gure 5.4a.

2. Place the discharge tubing into a waste carboy located LOWER than the pump inlet.

Fig. 5-4a

Fig. 5-4b

3. Begin siphon with half strokes until liquid starts to ow. Leave shaft fully extended as shown in gure 5.4b to allow siphon to operate.

4. To stop siphon, depress handle and remove pump from tank.

www.cbsscientic.com

SECTION 6 Recommended Buffers and Reagents

Warning: Do not mix buffer in the DGGE tank!

6.1 Stock Solutions Acrylamide Stock Solution 40% Acrylamide/Bis (37.5:1)

For 100ml use the following:

38.93 g of acrylamide

1.07 g of Bis-acrylamide

Add dH2O (or RO-H2O) to 100ml. Do not autoclave

50x TAE Gel Running Buffer

For 1 liter use the following: 242g Tris Base

57.1ml Glacial acetic acid 100ml .5 M EDTA, pH 8.0

Add dH2O (or RO-H2O) to 1 liter

Denaturant Stock Solution (7.5% gel)

For 100ml use:

Percentage 10 20 30 40 50 60 70 80 90 100

40% Acrylamide/

Bis (ml)

50% TAE Buffer

(ml)

Formamide (ml) 4 8 12 16 20 24 28 32 36 40

Urea (g)

18.8 18.8 18.8 18.8 18.8 18.8 18.8 18.8 18.8 18.8

2 2 2 2 2 2 2 2 2 2

4.2 8.4 12.6 16.8 21 25.2 29.4 33.6 37.8 42

Add dH20 (or RO-H2O) to 100ml

Store denaturant stock solutions at 4º C in amber bottles

Polymerization Catalysts:

1. TEMED (10ul/ml gel solution)

2. APS Stock Solution (.6ul/ml gel solution) For 50 ml of APS stock (10%) use 5 g of ammonium persulphate and water to 50ml. Store at 4ºC.

Neutral Loading Solution

For preparation of 10ml: 20% Ficoll or sucrose, 2 g of Ficoll 400 or sucrose 10mM Tris-HCl, pH 7.8, 100ul of 1.0 M Tris-HCl, pH 7.8 1mM EDTA, 20 ul of 0.5 M EDTA, pH 8.0

0.1% dye (BPB), 10 mg of dye Water to 10ml

Cipher DGGE Instructions 2/28/14

SECTION 7 Maximum Well/Comb Volumes

Vertical Comb Options, short wells, overall length = 13.1cm

Cat. # Tooth depth

(mm)

VGC-0716-177 10 16 0.75 5.2 29 VGC-0720-177 10 20 0.75 4.2 23 VGC-0722-177 10 22 0.75 4.35 25 VGC-0729-177 10 29 0.75 2.9 16 VGC-0730-177 10 30 0.75 2.8 16 VGC-1016-177 10 16 1.0 5.2 39 VGC-1020-177 10 20 1.0 4.2 32 VGC-1022-177 10 22 1.0 4.35 32 VGC-1029-177 10 29 1.0 2.9 22 VGC-1030-177 10 30 1.0 2.8 21 VGC-1516-177 10 16 1.5 5.2 58 VGC-1520-177 10 20 1.5 4.2 47 VGC-1522-177 10 22 1.5 4.35 49 VGC-1529-177 10 29 1.5 2.9 33 VGC-1530-177 10 30 1.5 2.8 32

Vertical Comb Options, deeper wells, overall length = 13.1cm

Cat. # Tooth Depth

(mm)

VGC-0716 19.1 16 0.75 5.2 55 VGC-0720 17.7 20 0.75 4.2 42 VGC-0722 17.7 22 0.75 4.35 25 VGC-0729 17.7 29 0.75 2.9 29 VGC-0730 17.7 30 0.75 2.8 28 VGC-1016 19.1 16 1.0 5.2 74 VGC-1020 19.1 20 1.0 4.2 55 VGC-1022 17.7 22 1.0 4.35 57 VGC-1029 17.7 29 1.0 2.9 38 VGC-1030 17.7 30 1.0 2.8 37 VGC-1516 19.1 16 1.5 5.2 110 VGC-1520 17.7 20 1.5 4.2 83 VGC-1522 17.7 22 1.5 4.35 85 VGC-1529 17.7 29 1.5 2.9 58 VGC-1530 17.7 30 1.5 2.8 56

# of

Teeth

# of

Teeth

Thickness

of Teeth

Thickness

of Teeth

Width of

Teeth

Width of

Teeth

Recommended

Max. Sample

Vol./Well

Recommended

(µl)*

Max. Sample

Vol./Well

(µl)*

Maximum loading volume is calculated by taking 75% of the total tooth volume.

www.cbsscientic.com

SECTION 8 References

1. S.G. Fischer and L.S. Lerman, (1983) PNAS 80:1579.

2. R.M. Myers, T. Maniatis, L.S. Lerman, (1987) Methods in Enzymology, 155: 501-529.

3. R.M. Meyers, V.C. Shefeld, D.R. Cox (1988) “Genome Analysis: A Practical Approach,” Ed. K Davies, IRL Press, Oxford. PP. 95-13

4. V.C. Shefeld, D.R. Cox, L.S. Lerman and R.M. Meyers, (1989) PNAS, 86:232-236

5. Guldberg, P., Henriksen, K.F., and Guttler, F. (1993). “Molecular analysis of phenylke-“Molecular analysis of phenylke-

tonia in Denmark: 99% of the mutations detected by denaturing gradient gel electrophoresis.” Genomics 17:141-146

6. Moyret, C., Thellet, C., Puig, P.L., Moles, J-P., Thomas, G. and Hamelin, R. (1994).

“Relative efciency of denaturing gradient gel electrophoresis and single-strand

conformation polymorphisms in the detection of mutations in exons 5 to 8 of the p53 gene.” Oncogene 9:1739-1743

7. Shefeld, V.C., Beck, J.S., Kwitek, A.E., Sandstrom, D.W., and Stone, E.M. (1993). “The sensitivity of single-strand conformation polymorphism analysis for the detection

of single bas substitutions.” Genomics 16: 325-332.

8. Abrams, E.S. Stanton, V.P. Jr., (1992) “Use of Denaturing Gradient Gel Electrophoresis to Study Conformational Transitions in Nucleic Acids” Methods in Enzymology

212:71-104.

Blog: http://ddgehelp.blogspot.com/2005/11/updated-dgge-guide.html

Cipher DGGE Instructions 2/28/14

SECTION 9 Troubleshooting

9.1 Unit Will Not Operate (no heat, cooling, or pumping)

• Check that the power cord is plugged in to an operating electrical outlet.

• Check that the Circuit Breaker/Power Switch is ON.

• Check that the Controller Power Switch is ON.

• Check that the Safety Set temperature is higher than the control temperature set

point.

• Turn power off, press the Safety Set Reset button, and restore power.

9.2 No Pumping

• Check the uid level of the bath to be sure the pump head is covered with uid.

• If the pump motor does not spin, verify that the Pump Speed Switch is either in the HIGH or LOW position. If it is in the middle, the pump will not receive power.

• Check the pump impeller for obstructions (it should turn freely).

9.3 Slow or Insufcient Pumping

• Check for low line voltage, particularly when the heater is on.

9.4 No Heating

• Verify that the unit is pumping properly.

• If the heat light is not lit, check the control temperature set point and bath temperature to verify that heating is required.

• Verify that the liquid in the bath covers the heater coils.

• Check that the Software High Limit value has not been exceeded (FLt 1 appears on the display).

9.5 Insufcient Heating

• Verify that the unit is pumping properly.

• Check for proper line voltage.

• Check for excessive external cooling load on unit.

• At higher temperatures, insufcient heating could be due to excessive heat loss from tanks, hoses, or vapor from the tank.

• Check that the heat demand from connected devices or experiments does not exceed the heating capabilities of the unit.

• Allow sufcient time for bath temperature to stabilize when changes in heat load or set point are made.

9.6 Triac Failure

• (FLt4) appears on the display, indicating that the heater triac has failed or line supply voltage has a source of extreme interference from other equipment. Plug the unit into another power source. If it still displays triac failure, a triac or triac driver needs replacement.

9.7 Samples Not Entering Gel After Loading

• When running one gel on a dual cassette, the conversion plate must be clamped to the unused side to complete the cathode buffer chamber.

www.cbsscientic.com

SECTION 10 Maintenance of Equipment

10.1 Care and Handling

The plastic components of the DGGE Systems 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 gel cassettes, 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 nonabrasive 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 undiluted alcohol's.

• 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 sufcient after a thorough wash.

10.2 Maintenance

The following inspection and maintenance procedures will help maintain the safety and reliable performance of the Cipher DGGEK systems. Replacement parts can be ordered by calling 858-755-4959 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.

Cipher DGGE Instructions 2/28/14

SECTION 11 Ordering Information

CAT. # DESCRIPTION

CIPHER DGGE ELECTROPHORESIS SYSTEMS DGGEK-1001

DGGEK-2001

DGGEK-2401

DGGEK-4801

C.B.S. Scientific DGGEK-2401 User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual