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- Página 1 Hoefer HE-PLUS Horizontal Gel Electrophoresis System HE-PLUS/Rev. B0/07-12...
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Página 2: Tabla De Contenido
Contents Important Information ..........ii Waste Electrical and Electronic Equipment (WEEE) .......vii Specifications ............ix Operating Instructions ..........1 Troubleshooting Guide ..........11 Solutions ............12 References ............13 Ordering Information ..........14 Care and Maintenance .........15 • pi... -
Página 3: Important Information
Important Information – English • Tento nástroj je určen v souladu s EN61010-1:2001 elektrické bezpečnostní normy. Přesto, že by měly být použity pouze řádně vyškolený operátorů. Číst celé • If this equipment is used in a manner not specified by toto ruční před použitím nástroje a použití pouze v Hoefer, Inc. the protection provided by the equipment souladu s pokyny. may be impaired. • Přístroj musí být vždy používají se na výkonu zemi vést • This instrument is designed for indoor laboratory šňůra správně zemněny k zemi na síti výústce. use only. • Využití pouze nepoškozené elektrické dráty a vybavení • Only accessories and parts approved or supplied by pro napětí budete používat. Všechna zařízení spojené Hoefer, Inc. may be used for operating, maintaining, s vysokým napětím by měla být v souladu s EN61010- and servicing this product. 1:2001. • Warning! Because this instrument can develop suffi- • Si ponechá nástroje jako suchý a čistý jako možné. cient voltage and current to produce a lethal shock, Otřete pravidelně s a měkké, vlhkým hadříkem. Nechť care must be exercised in its operation. je nástroj nenastavený úplně před použitím. • This instrument is designed in accordance with the • Nejsou provozována na nástroj v extrémní vlhkost (nad EN61010-1:2001 electrical safety standard. Never- 80%). Předešlo kondenzaci o pronájmu jednotky na theless, it should be used only by properly trained okolní teplotu nechá při přijímání nástroj z chladnější... - Página 4 80%). Undgå kondensation ved lade enheden equili- Hoeferille, Inc. suojelu ehkäisty varusteille saattaa brate til omgivende temperatur ved tageen instru- olla avuton. mentets fra et koldere til et varmere miljø. • Tämä väline suunnitellaan sisälaboratoriokäytölle vain. • At tillade tilstrækkelig afkøling, forsikrer, at lufthullerne af instrumentet er ikke dækket. • Vain lisävarusteet ja osat hyväksyivät tai toimitti Hoeferin oheen, Inc.:ää voi käyttää käyttämiselle, Belangrijke Informatie – Dutch valvoalle, ja servicing tämä tuote. • Varoittaminen! Koska tämä väline voi kehittää riittävä • Indien deze uitrusting in een manier wordt gebruikt jännitteen ja virran tuottaa kuolettavan järkytyksen, die niet door Hoefer is gespecificeerd, Nv. de huolta täytyy harjoittaa toiminnossaan. bescherming die door de uitrusting is verzorgd kan • Tämä väline suunnitellaan EN61010-1:2001 sähkö- worden geschaad. turvallisuusstandardin mukaisesti. Silti pitäisi käyt- • Dit instrument is voor binnenlaboratoriumgebruik tää vain ohi oikeasti koulutetut käyttäjät. Lue tämä enkel ontworpen. kokonainen manuaalinen ennen välinettä ja käyttö vain ohjeiden mukaan. • Enkel onderdelen en delen keurden goed of leverden door Hoefer, Nv. kan voor het bedienen worden • Välinettä täytyy käyttää aina valtanuoran maalyijystä gebruikt, handhavend en onderhouden van dit perusti oikein maadoittaa sähköverkkoaukossa.
- Página 5 • Um das genügend Abkühlen zu erlauben, stellen Sie • Ne pas fonctionner l’instrument dans l’extrême sicher, dass die Öffnungen des Instrumentes nicht humidité (au-dessus de 80%). Eviter la condensa- bedeckt werden. tion en laissant l’equilibrate d’unité à la température ambiante en prenant l’instrument d’un plus froid à un Informazioni Importanti – Italiano environnement plus chaud. • Se quest’apparecchiatura è usata in un modo • Permettre le refroidissement suffisant, garantir que les specificato da Hoefer, Inc. la protezione fornito conduits de l’instrument ne sont pas couverts. dall’apparecchiatura potrebbe essere indebolita. Wichtige Informationen – German • Questo strumento è disegnato per l’uso di laboratorio interno solo. • Wenn diese Ausrüstung gewissermaßen nicht angege- • Solo gli accessori e le parti hanno approvato o hanno ben durch Hoefer, Inc verwendet wird, kann der fornito da Hoefer, Inc. potrebbe essere usato per oper- durch die Ausrüstung zur Verfügung gestellte Schutz are, per mantenere, e per revisionare questo prodotto. verschlechtert werden. • Avvertendo! Perché questo strumento può sviluppare • Dieses Instrument wird für den Innenlaborgebrauch il voltaggio sufficiente e la corrente di produrre una nur dafür entworfen.
- Página 6 • Non opera lo strumento nell’umidità estrema (al di toriach kryty tylko. sopra di 80%). Evitare la condensazione lasciando • Tylko akcesoriów i części zatwierdzone lub dostarczone l’unità equilibra alla temperatura ambiente quando przez Hoefer, Inc. mogą być wykorzystane do eksp- portare lo strumento da un più freddo a un ambiente loatacji, utrzymania i obsługi tego produktu. più caldo. • Uwaga! Ponieważ ten akt prawny może być rozwinięcie • Di permettere raffreddare sufficiente, assicura che gli odpowiednich napięcie i bieżących do wyprodu- sbocchi dello strumento non sono coperti. kowania śmiertelnego szoku, opiekę musi być wykony- wane w działaniu. Viktig Informasjon – Norwegian • Ten instrument został zaprojektowany zgodnie z tym EN61010-1: 2001 Bezpieczeństwo elektryczne • Hvis dette utstyret blir brukt i en måte ikke spesifisert standard. Niemniej jednak, należy stosować jedynie ved Hoefer, Inc. beskyttelsen som ha blitt git av utsty- przez odpowiednio przeszkoleni operatorów. Znajdą ret kan bli svekket. państwo to cały podręcznika przed zastosowaniem • Dette instrumentet er utformet for innendørs labora- instrumentu i stosować jedynie zgodnie z instrukcjami. toriumbruk bare. • Instrument musi zawsze być wykorzystane z ziemi • Bare tilbehør og deler godkjente eller forsynte ved doprowadzić do zasilania detonującego właściwie Hoefer, Inc. kan bli brukt for drive, vedlikeholde, og uzasadnione na ziemię w sieci wodociągowej...
- Página 7 (encima de 80%). Evite condensación permitiendo la terra nos canos saída principais. unidad equilibra a la temperatura ambiente al tomar el instrumento de un más frío a un ambiente más tibio. • Usa fio eléctrico só intacto e equipamento específico para as voltagens que você usará. Todo equipamento • Permitir refrigeración suficiente, asegure que las conectado a voltagem alta deve ser de acordo com aberturas del EN61010-1:2001. Viktig Information – Swedish • Mantem o instrumento tão seco e limpo como possível. Limpe regularmente com um pano húmido • om denna utrustning används i ett sätt som inte har macio. Deixe o instrumento secar completamente specificeras av Hoefer, Inc. skyddet tillhandahöll vid antes de uso. utrustningen kan skadas. • Não opera o instrumento em humidade extrema • Detta instrument formges för inomhuslaboratorium (acima de 80%). Evite condensação deixando o equili- användning bara. brate de unidade a temperatura ambiental quando tomar o instrumento de um mais frio a um ambiente • Bara medhjälpare och delar godkände eller levererade mais quente. vid Hoefer, Inc. kan användas för fungera, underhålla, och servicing denna produkt. • Permitir esfriar suficiente, assegura que as aberturas do instrumento não são cobertas. • varna! Därför att detta instrument kan utveckla tillräck- lig spänning och ström att producera en dödlig stöt,...
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Página 8: Waste Electrical And Electronic Equipment (Weee)
Waste Electrical and Electronic Equipment (WEEE) English This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately. Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment. Ce symbole indique que les déchets relatifs à l’équipement French électrique et électronique ne doivent pas être jetés comme les ordures ménagères non-triées et doivent être collectés séparément. Contactez un représentant agréé du fabricant pour obtenir des informations sur la mise au rebut de votre équipement. German Dieses Symbol kennzeichnet elektrische und elektronische Geräte, die nicht mit dem gewöhnlichen, unsortierten Hausmüll entsorgt werden dürfen, sondern separat behandelt werden müssen. Bitte nehmen Sie Kontakt mit einem autorisierten Beauftragten des Herstellers auf, um Informationen hinsichtlich der Entsorgung Ihres Gerätes zu erhalten. Italian Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere raccolti separatamente. Per informazioni relative alle modalità di smantellamento delle apparecchiature fuori uso, contattare un rappresentante autorizzato del fabbricante. Spanish Este símbolo indica que el equipo eléctrico y electrónico no debe tirarse con los desechos domésticos y debe tratarse por separado. Contacte con el representante local del fabricante para obtener más información sobre la forma de desechar el equipo. Swedish Denna symbol anger att elektriska och elektroniska ... -
Página 9: Usage Guidance And Restrictions
Packing List Hoefer HE-PLUS Gel System Units include: Tank with lid, power supply, power cord, and standard casting set (one – 13 × 12.5 cm UV Transmittant Casting Tray, two – 6 × 12.5 cm UV Transmittant Casting Trays, four – 1 mm 14/28 reversible multichannel compatible combs, and a casting stand). The packing lists should be referred to as soon as the units are received to ensure that all components have been included. The unit should be checked for damage when received, save packaging for carrier. Please contact your supplier if there are any problems or missing items. Usage Guidance and Restrictions • Temperature range between 4 °C and 40 °C. • M aximum relative humidity 80% for temperatures up to 31 °C decreasing linearly to 50% relative humidity at 40 °C. • Not for outdoor use. This apparatus is rated POLLUTION DEGREE 2 in accordance with IEC 664. POLLUTION DEGREE 2, states that: “Normally only non-conductive pollution occurs. Occasionally, however, a temporary conductiv- ity caused by condensation must be expected”. • pviii... -
Página 10: Specifications
Specifications Unit dimensions (W × L × H) 24.5 × 17.0 × 6.2 cm Gel dimensions (W × L) 12.5 × 13.0 cm Maximum sample capacity 1 12 samples (4 combs, 28 samples each) Buffer capacity 350 ml Distance between electrodes 13.5 cm Electrophoresis Tank Overall dimension (W × L × H) 18.3 × 16.4 × 5.6 cm Material characteristic U V transmitting (50% at 254 nm, 80% at 312 nm) Solution volume 3 50 ml (includes buffer and gels) Safety Lid Overall dimension (W × L × H) 19.7 × 16.9 × 3.8 cm Material characteristic U V non-transmitting acrylic Power Supply Overall dimension (W × L × H) ... -
Página 11: Operating Instructions
Operating Instructions A. Guidelines for Selecting Electrophoresis Buffers and Gel Concentrations The two most commonly used buffers for hori- zontal electrophoresis of double stranded DNA in agarose gels are Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE). While the resolv- ing powers of these buffers are very similar, the relative buffer capacities are very different, conferring different run attributes which are summarized below: Tris-acetate has traditionally been the more ... - Página 12 Make 500 ml of either 1X TAE or 1X TBE electrophoresis buffer. Weigh an appropriate quantity of agarose (see Table 1) and place it into a 250 ml flask. Add a sufficient quantity of either 1X TAE or 1X TBE buffer (prepared in step 1) to achieve a final volume of 100 ml agarose solution. Table 1: Gel Concentrations and Resolving Ranges Concentration Agarose (g) Efficient Range of Agarose in per 100 ml of Separation Gel (%w/V) Buffer of Linear DNA (Kb) 0.3 0.3 5 – 60 0.6 0.6 1 – 20 0.7 0.7 0.8 – 10 0.9 0.9 0.5 – 7 1.2 1.2 0.4 – 6 1.5 1.5 0.2 – 3 2.0 2.0 0.1 – 2 Table taken from Sambrook, J., Fritsch, E.F., & Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 1, 6.8 613.
- Página 13 Make note of the total solution volume so that degree of evaporation can be determined and corrected for. Heat the agarose slurry in a microwave oven for 90 seconds. Swirl the flask to make sure any grains sticking to the walls enter into the solution. Undissolved agarose appears as small “lenses” floating in the solution. Heat for an additional 30 – 60 seconds. Re-examine the solution and repeat the heating process until the agarose completely dissolves. Add deionized water to replace any volume lost through evaporation during the heating process. Note: The addition of Ethidium Add your detection reagent (i.e. Ethidium Bromide) to Bromide to both the gel and the manufacturers’ recommended concentration. Mix the running buffer will result in maximum detection levels by by gently swirling the flask. providing high levels of sample fluorescence with an evenly low level of background. • p3...
- Página 14 B. Casting the Gel Place the gel casting stand on a lab bench. Insert the gel casting tray into the casting stand. If you are using the 6 × 12.5 cm gels place the spacer in the center of the casting stand, then insert the two 6 × 12.5 cm landscape gel trays. When the gel solution has cooled to approximately 55 °C, slowly pour it into the gel tray. If hotter gel solutions are routinely poured, the tray may warp over time. If bubbles form on the surface of the gel upon pouring, use the comb to either pop them or lightly brush them to the sides of the gel. If large bubbles are allowed to harden within the gel, they may cause artifacts to occur during electrophoresis. • p4...
- Página 15 Insert one or more combs by placing them into the slots in the casting stand. For best results, place the comb in the slot nearest the end of the casting fixture. If two combs are desired, place the second in the center comb slot. Comb Well Sample Description Width Vol. 1 mm Code No. 1 mm, 14 tooth 5 mm 5 µl HE-PLUS1428 dual format 1 mm, 28 tooth 2.5 mm 2.5 µl HE-PLUS1428 dual format 1 mm, 5 tooth 8 mm 8 µl HE-PLUS0508 dual format 1 mm, 8 tooth 4 mm 4 µl HE-PLUS0508 dual format Allow the gel to harden undisturbed for at least 30 minutes. • p5...
- Página 16 C. Removing the Comb When the gel is solidified and fully opaque, carefully remove the comb with a gentle wiggling, upward motion. If the comb is difficult to remove or if a low percentage gel is being used, overlay the comb area with a small volume of 1X electrophoresis buffer to preserve the integrity of the wells. Check the wells to ensure their bases are intact. D. Loading the Samples onto the Gel Remove the casting tray containing the hardened agarose gel from the casting fixture by lifting the ends. Place the tray and gel into the main unit assembly such that the sample wells are on the same end as the negative (black) electrode. Note: Use of the same batch of Fill the unit with the remaining 1X electrophoresis electrophoresis buffer for both buffer containing Ethidium Bromide made previously, the gel and the running buffer is very important. Slight variations covering the gel to a depth of 1– 5 mm. Approximately in buffer composition between 350 ml of buffer will be required. gel and running buffer may result in ionic or pH gradients that can significantly impact the mobility of the samples. Load the samples into the wells with a micropipette or similar device taking care not to puncture the bottom of the wells or load the sample onto the top of the gel. • p6...
- Página 17 E. Electrical Connections to the Safety Lid The Hoefer HE-PLUS Gel System can only be operated with the safety lid in place. Elec- trical current is supplied through the tank electrodes from the power supply. A micro switch connector in the power supply ensures a complete current path, yet allows the lid to be removed from the unit without disturbing the loaded samples.
- Página 18 F. Setting the Power Supply Ensure an approved power cord that satisfies your regional voltage standard is used. Input voltage is automatically detected by the system. Use the mode key > to move between voltage, amperage and time parameters. To increase or decrease voltage use up (^) and down (v) arrow keys. To increase or decrease amperage use up and down arrow keys. Set the timer. Increase or decrease the value with the up and down arrow keys. Between 99 hours and 59 minutes can be set as the run time. Set “0” for Continuous. On the left side of the Output key the flashing LED indicates that the timer operation has been paused. When setting up the timer in this state, set up after having pushed the output button for a long time so as to reset the timer. Select the required output voltage up to 150 volt or 400 mA. Press the start/stop key to start the run. • p8...
- Página 19 To pause the run select Pause. During the pause mode the voltage amperage or time can be changed by highlighting the function and using the arrow keys then pressing enter. Once the changes have been made the CAUTION: Do not jar or bump the gel box once the lid is place. start button can be pressed to resume the run. The electrical connection is made by gravity once the lid is in position. While this design helps To stop the run press the pause button for 30 seconds. to minimize sample disturbance Stop will appear. during lid placement, it also may result in a disruption of power to the unit if the lid or unit are disturbed during the run. H. Sample Electrophoresis The maximum suggested applied voltage for the electrophoresis of DNA in agarose gels using the Hoefer HE-PLUS Gel System is 150 volts. In a 1% TBE gel, this translates into a run time of approximately 1 hour. Lower voltages may be used, CAUTION: DO NOT EXCEED THE of course, and as a general rule, a 70 V run will take MAXIMUM OPERATING VOLTAGE twice as long as a 145 V run. Higher voltages may OF 150 VOLTS. be used to decrease run time, however, if the unit is being operated at higher voltages than 150 V, the heat generated during electrophoresis may decrease sample resolution. Such artifacts may be avoided by running the unit in a cold room or adding 1X electrophoresis buffer “ice cubes” to keep the unit properly cooled. Follow the sample migration into the gel using the loading dye as an indicator. (See page 12 for the Sample Loading Buffer recipe.) Allow the samples to migrate until the fragments have separated, normally until the bromophenol blue dye front has migrated ¾ of the way down the gel.
- Página 20 I. Detection and Documentation of Separated Fragments At the completion of the run, turn off the power supply and disconnect the power cord. Remove the lid and remove the gel tray. Alternatively the entire tank can be placed on a Transilluminator. Note: If the gel contains Ethidium Ethidium Bromide stained samples are visualized by Bromide, the progress of exposing them to medium wavelength (312 nm) UV electrophoresis may be monitored during the run by turning off the light. Because the gel casting tray is UV transmittant, power supply, removing the lid, the gel does not need to be removed from the tray and shining a medium-wave UV before viewing. Place the gel casting tray containing light onto the gel. The resolved the gel on the filter surface of a UV Transilluminator bands will appear as orange bands against a dark purple background. for convenient viewing. • p10...
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Página 21: Troubleshooting Guide
Troubleshooting Guide Problem Cause Solution The LCD screen remains AC power cord is not Check AC power cord connections at both ends. blank and the fan does connected. Use the correct cords. not run when the power is turned on. The fuse has blown. Replace the fuse. Operation stops with Electrophoresis tank Check the connections to the power supply and alarm: The screen is not connected to on your electrophoresis cell to make sure the displays “NO LOAD”. the power supply connection is intact; check condition of wires or there is a broken in electrophoresis unit. Close the circuit by circuit in the reconnecting the cables. Press RUN/PAUSE to electrophoresis cell. restart the run. Buffer concentration Replace buffer. incorrect. Operation stops with Circuit is interrupted. Verify that the running buffer is correct. alarm: Display shows Verify the all connections are attached correctly. “OVER VOLTAGE”. Turn the Power Switch off and on again; restart application. If you cannot restart the instrument, turn off ... -
Página 22: Solutions
Solutions Tris Acetate EDTA Buffer (TAE) 1X Working Concentration: 40 mM Tris base 20 mM Glacial Acetic Acid (NaOAc) 2.0 mM EDTA pH 8.3 10X Stock Solution: 48.4 g Tris Base 16.4 g or 11.42 ml NaOAc 7.4 g EDTA or 20 ml 0.5 M EDTA (pH 8.0) O to 1 liter Tris Borate EDTA Buffer (TBE) 1X Working Concentration: 89 mM Tris Base 89 mM Boric Acid 2.0 mM EDTA pH 8.0 10X Stock Solution: 108 g ... -
Página 23: References
References 1. Lehrach, H., et al. 1977. Biochemistry 16:4743. 2. Sambrook, J., Fritsch, E.F., and Maniatis, T., (1989). Molecular Cloning, A Laboratory Manual, vol 1. Cold Spring Harbor Press, New York. 3. Selden, R.F. (1988) Analysis of RNA by Northern Hybridization,” in Current Proto- cols in Molecular Biology, F.M. -
Página 24: Ordering Information
Ordering Information Product Code No. HE-PLUS Electrophoresis HE-PLUS-115V complete system 115 V. Includes gel tank, safety lid, power supply and standard casting kit. H E-PLUS Electrophoresis HE-PLUS-230V complete system 230 V. Includes gel tank, safety lid, power supply and standard casting kit. Accessory Items H E-PLUS Large Gel trays HE-PLUS11 12.5 × 13 cm set of 2 H E-PLUS Medium Gel trays HE-PLUS12 12.5 × 6 cm set of 2 H E-PLUS Mini Gel trays HE-PLUS13 6 × 6 cm set of 4 H E-PLUS Large/Medium comb HE-PLUS1428 14/28 Wells set of 2 H E-PLUS Mini comb ... -
Página 25: Care And Maintenance
Care and Maintenance Cleaning Units are best cleaned using warm water and a mild detergent. Water at temperatures above 60 °C can cause damage to the unit and components. The units Important: The units should should not be left in detergents for more than never come into contact with the following cleaning agents, 30 minutes. The tank should be thoroughly rinsed these will cause irreversible and with warm water and distilled water to prevent build cumulative damage: up of salts but care should be taken not to damage Acetone, Phenol, Chloroform, the enclosed electrode. Vigorous cleaning is not Carbon tetrachloride, Methanol, necessary or advised. Air drying is recommended Ethanol, Isopropyl alcohol, Alkalis. before use. RNase Decontamination This can be performed using the following protocol: • C lean the units with a mild detergent as described above. • W ash with 3% hydrogen peroxide (H ) for 10 minutes. • R inse with 0.1% DEPC- (diethyl pyrocarbonate) treated distilled water. • C aution! DEPC is a suspected carcinogen. Always wear gloves and safety glasses. ™ RNaseZAP (Ambion) can also be used. Please ... - Página 26 Hoefer, Inc. 84 October Hill Road Holliston, MA 01746 Toll Free: 1-800-227-4750 Phone: 1-508-893-8999 Fax: 1-508-893-0176 E-mail: support@hoeferinc.com Web: www.hoeferinc.com Hoefer is a registered trademark of Hoefer, Inc. RNaseZAP is a registered trademark of Ambion. © 2012 Hoefer, Inc. — All rights reserved. Printed in the USA.