HE99X User Manual – English
Contents
1. Nejsou provozov na s pufru teplot ch nad maxim ln stanovenou technick mi specifikacemi P eh t zp sob nenapraviteln po kozen jednotka lt igtig Information Danish Hvis dette udstyr bruges i en m de ikke specificeret ved Hoefer Inc den beskyttelse som er blevet forsynet af udstyret kan m ske svaekkes Dette instrument er designet for indendors laboratoriumbrug bare Bare tilbehor og del godkendede eller forsynede ved Hoefer Inc kan m ske bruges for drive funktionsfejl og betjening dette produkt bruger Bare en stramforsyning der er CE markerede eller sikkerhed som er blevet attesteret af en som nationalt er blevet anerkendt prove laboratorium Sikkerhedl get m vaere p plads for forbinding stramforsyningsblyet til en stremforsyning Drejer alle stramforsyningskontroller af og afbryder kraftblyet for fjerning sikkerhedl get Cirkulerer bare vand eller 50 50 vand ethylene glykol gennem varmeveksleren i s fald udrustet Forbind ikke varmeveksleren til en vandhane eller nogen kolemiddelkilde hvor vandtrykket er unregulated Introducerer Aldrig antifreeze eller noget organisk opl sningsmiddel ind i nogen del af instrumentet Organiske opl sningsmidler vil for rsage uboelig skade til enheden Driver ikke med st dpudetemperaturer over maksimummet specificerede tekniske specifications Overheding vil for rsage uboelig skade til enheden e pii Belangrijke Informatie2. Bezpiecze stwo lid musi by w miejsce przed pod czeniem zasilania prowadzi do zasilania Za wszystkie r d a zasilania urz dzenia steruj ce off i od czy moc prowadzi przed odbiorem bezpiecze stwa lid Kr tylko wody lub wody 50 50 ethylene glycol wymiennik ciep a poprzez je li tak wyposa one Nie nale y po czy wymiennik ciep a woda z kranu lub jakimkolwiek ch odziwo r d a je eli ci nienie wody jest nieuregulowanych Nigdy nie wprowadza rozpuszczalnika organicznego przeciw zamarzaniu lub jakichkolwiek na dowoln cz dokumentu Rozpuszczalniki organiczne spowoduje nieodwracalne szkody dla jednostki Nie dzia aj w buforze temperatury powy ej maksymalnego okre lone specyfikacje techniczne Przegrzania spowoduje nieodwracalne szkody dla jednostki Informa es Importantes Portuguese Se este equipamento usado numa maneira n o especificada por Hoefer Inc que a protecc o fornecida pelo equipamento pode ser comprometida Este instrumento projectado para uso de interior de laborat rio s S acess rios e partes aprovaram ou forneceu por Hoefer Inc pode ser usada para operar manter e servicing este produto S usa um estoque de poder que CE marcou ou seguranca registrada por um nacionalmente reconhecido testando laborat rio A tampa de seguran a deve estar em lugar antes de ligar o estoque de poder leva a um estoque de poder Desliga todos cont
3. Dutch Indien deze uitrusting in een manier wordt gebruikt die niet door Hoefer Inc is gespecificeerd de bescherming die door de uitrusting is verzorgd kan worden geschaad Dit instrument is voor binnenlaboratoriumgebruik enkel ontworpen Enkel onderdelen en delen keurden goed of leverden door Hoefer Inc kan voor het bedienen worden gebruikt handhavend en onderhouden van dit product gebruik Enkel een netvoeding die CE is markeerde of veiligheid die door een is gecertificeerd die nationaal is herkend testene laboratorium Het veiligheidsdeksel moet in plaats voor het verbinden van de netvoeding leidt tot een netvoeding zijn Doe alle netvoedingscontroles Uit en koppel los de machtleiding voor het verwijderen van het veiligheidsdeksel Circuleer enkel water of 50 50 water ethyleenglycol door de hitte exchanger zo ja uitrust Verbind de hitte exchanger naar een waterkraan of koelmiddelbron niet waar de waterdruk niet geregulariseerd is Stel Nooit antivriesmiddel of organische oplosmiddelen in deel van het instrument voor Organische oplosmiddelen zullen onherstelbare schade aan de eenheid veroorzaken Bedien niet met buffertemperaturen boven het maximum specificeerde technische specificaties Oververhittend zal onherstelbare schade aan de eenheid veroorzaken T rke Tietoa Finnish Jos t t varusteita k ytet n tavassa ei m ritetty Hoefer Inc suojelu ehk isty varusteille saat
4. manual Hoefer HE99X Submarine electrophoresis unit um HE99X IM Rev LO 07 12 Hoefer Contents Important Information eee ii Waste Electrical and Electronic Equipment WEEE ee vii Submarine Electrophoresis Unit Function and description iicet denia 1 e ele EE 2 Operating instructions 3 Care and maintenance cccccnnnncncncnnnnanononononanininnns 8 Troubleshootllg ose A O AWA AW ARR sea d iren 9 Buffers volumes and notes 2 2002 10 Loading buffer and sample volumes 12 Ordering information 16 Important Information English If this equipment is used in a manner not specified by Hoefer Inc the protection provided by the equipment may be impaired This instrument is designed for indoor laboratory use only Only accessories and parts approved or supplied by Hoefer Inc may be used for operating maintaining and servicing this product Only use a power supply that is CE marked or safety certified by a nationally recognized testing laboratory The safety lid must be in place before connecting the power supply leads to a power supply Turn all power supply controls off and disconnect the power leads before removing the safety lid Circulate only water or 50 50 water ethylene glycol through the heat exchanger if so equipped Do not connect the heat exchanger to a water tap or any coolant source where the water pressure is unregulated Ne
5. 2 on handles orient the running tray on the running platform The comb back fits into any set of slots the comb back assembly shown in Fig 3 is oriented to fit into this slot UVT gel running tray 15 x 20cm foam pads 2 gel casting tray 15 x 20cm Da Fig 3 Assembled comb comb back fi L je to screws Seat the running tray between the foam pads in the casting tray by placing one end of the tray against the foam pad slightly compressing it then seating the other end of the tray against the opposite foam pad See arrows A and B in Fig 2 The running tray should lay flush against the bottom of the casting tray e Place the casting tray assembly on a leveling surface and level using the spirit level on the running tray as a guide Check that the comb assembly leaves 1 mm of space between the comb bottom and the running tray Remove the level and the comb assembly Prepare the combs o Align the two slots in the comb with the loosened thumb screws of the comb back Tighten the screws until the comb is just supported o Place the comb assembly into a set of slots on the running tray seated in the casting tray Adjust the comb so that the bottom of the teeth are z1 0 mm from the running tray Tighten the screws to secure the comb To run twice as many samples on the 15 and 20cm trays prepare two comb assemblies and place one near the cathode end
6. HE91A P 3 0 1 2 113 10 3 0 342 29 0 HE91A 10 1 5 10 9 7 15 14 5 HE91A 10 3 0 10 9 7 3 0 30 0 HE91A 15 1 0 15 ch 1 0 11 HE91A 15 1 5 15 Tel 15 10 6 HE91A 15 3 0 15 LL 3 0 21 3 HE91A 20 1 0 20 4 7 1 0 4 7 HE91A 20 1 5 20 4 7 15 11 HE91A 20 3 0 20 4 7 3 0 142 HE91A 30 1 0 30 3 0 1 0 3 0 Preparative combs form two reference wells for MW standards one on each side of the preparative well The first number is sample volume mm depth in the preparative well the second is volume mm in the reference well Agarose gel electrophoresis notes Agarose gel electrophoresis can be used to separate DNA fragments down to 0 1 kb or less Polyacrylamide gels are typically used for fragments smaller than 1 kb DNA mobility The suggested agarose concentration for separating fragments of various sizes is listed below Other factors affecting separation results include the selected running buffer the voltage setting the temperature and the presence of ethidium bromide Agarose concentrations for separating DNA fragments of various sizes effective range of resolution agarose of linear DNA fragments kb 0 5 1 to 30 0 7 0 8 to 12 1 0 0 5 to 10 1 2 0 4 to 7 1 5 0 2 to 3 Current Protocols in Molecular Biology p 2 5 2 1993 A common standard is a Hind III digest of lambda phage which gives eight fragments rang ing in size from 0 1 to 23 kb The bands are well resolved when run 2 hours on a 20 cm long 1 agarose gel i
7. lla d r vattnet trycket r unregulated Inf r aldrig kylv tska eller n got organiska l sningsmedel in i n gon del av instrumentet Organiskt l sningsmedel ska orsaka irreparable skada till enheten Anv nd inte med buffert temperaturer ver det h gsta angivna tekniska specifikationerna verhettning skulle orsaka irreparabla skador p enheten e pvi English EXE French R German R Ea Italian R ml Spanish 14 Swedish R gum Waste Electrical and Electronic Equipment WEEE 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 d chets relatifs l quipement lectrique et lectronique ne doivent pas tre jet s comme les ordures m nag res non tri es et doivent tre collect s s par ment Contactez un repr sentant agr du fabricant pour obtenir des informations sur la mise au rebut de votre quipement Dieses Symbol kennzeichnet elektrische und elektronische Ger te die nicht mit dem gew hnlichen unsortierten Hausm ll entsorgt werden d rfen sondern separat behandelt werden m ssen Bitte nehmen Sie Kontakt mit einem autorisierten Beauftragten des Herstellers auf um Informationen hinsichtlich der Entsorgung Ihres Ger
8. tes zu erhalten Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere raccolti separatamente Per informazioni relative alle modalit di smantellamento delle apparecchiature fuori uso contattare un rappresentante autorizzato del fabbricante Este s mbolo indica que el equipo el ctrico y electr nico no debe tirarse con los desechos dom sticos y debe tratarse por separado Contacte con el representante local del fabricante para obtener m s informaci n sobre la forma de desechar el equipo Denna symbol anger att elektriska och elektroniska utrustningar inte f r avyttras som osorterat hush llsavfall och m ste samlas in separat Var god kontakta en auktoriserad tillverkarrepresentant f r information ang ende avyttring av utrustningen e pvii Submarine Electrophoresis Unit Function and description The Hoefer HE99X unit electrophoretically separates nucleic acid fragments in a submarine gel The gel is first cast in a gel caster which is available in three lengths Once the gel sets the running tray is transferred to the platform of the electrophoresis unit and the gel is submerged under running buffer Fig 1 Horizontal submarine unit Color coded leads connect main components Gel casting kits combs and comb backs may be ordered separately Fig 2 illustrates a casting kit and
9. the ordering section tabulates all comb sizes and accessories electrodes in the unit base to the power supply Rest thumbs on both posts protruding through each end of lid while lifting both tabs on lid for easy lid removal electrode post 2 running platform leveling feet 4 chamber This declaration of conformity is only valid for the instrument when it is used in laboratory locations used as delivered from Hoefer Inc except for alterations described in the user manual and connected to other CE labeled instruments or products recommended or approved by Hoefer Inc Unpacking Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact your local Hoefer Inc sales office Inspect all components for damage that may have occurred while the unit was in transit If any part appears damaged contact the carrier immediately Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit Specifications Max voltage 200V Max wattage 20W Max amperage 100 mA Max operating temp 45 C Max buffer volume 1 2 liters Gel size 15cm wide x 10 15 or 20 cm long Environmental operating conditions Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Installation category II Pollution degree 2 Dimensions w x I x d 18 2 x 36 x 14cm i
10. Molecular Biology Greene Publishing and Wiley Interscience New York 1993 Sambrook J and Russell D W Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press 2001 e pl5 Ordering information product quantity code number HE99X Horizontal Agarose Submarine Unit complete 1 HE99X 15 1 5 Includes basic unit 15 x 20cm gel casting kit one 1 5 mm thick 15 well comb and comb back HE99X Horizontal Agarose Submarine Unit basic 1 HE99X Includes spirit level Order gel casting kit comb and comb back separately Accessories and replacement parts Buffer chamber assembly only 1 HE90X Comb back for HE99X comb with 2 screws 1 HE91 BK Lid with power cables 1 HE96X High voltage leads set 1 SE6056 HV Mylar sealing tape 1 roll 66 mm 1 SE1510 Foam sealing gaskets 4 HE98X Leveling feet 4 HE99XRK 1 Companion products MacroVue UV 20 Transilluminator 115 V 1 UV20 115V MacroVue UV 20 Transilluminator 230 V 1 UV20 230V HE99X gel casting kits Each size includes 1 gel casting tray 1 UVT running tray and 4 foam sealing gaskets 15 x 10cm 1 HE97X 10 15 x 15cm 1 HE97X 15 15x 20cm l HE97X 20 HE99X gel casting trays 15 x 10cm 1 HE95X 10 15 x 15cm 1 HE95X 15 15 x 20cm 1 HE95X 20 HE99X gel running trays 15 x 10cm 1 HE92X 10 15 x 15cm 1 HE92X 15 15 x 20cm 1 HE92X 20 e p16 Combs comb thickness well width no
11. bruker Bare en kraftforsyning som er CE merket eller sikkerhet som ha blitt sertifisert av et som e piv nasjonalt ha blitt anerkjent prover laboratorium Sikkerheten lokket m v re p plass for forbinding kraftforsyningene blyene til en kraftforsyning Vender all kraftforsyningsstyring av og frakopler kreftene blyene for fjerning sikkerheten lokket Sirkulerer bare vann eller 50 50 vann ethylene glykol gjennom oppvarmingen veksleren i s fall utstyrer Ikke forbind oppvarmingen veksleren til en vanntapp eller noe kjalemiddelkilde hvor vannet trykket er unregulated Introduserer Aldri antifreeze eller noe organisk losemiddel inn i noe del av instrumentet Organiske losemiddler vil for rsake irreparabel skade p enheten Driver med buffertemperaturer over maksimum ikke spesifiserte teknisk spesifikasjoner overoppheting vil for rsake irreparabel skade p enheten Wazne Informacje Polish Jezeli ten sprzet jest wykorzystywany w spos b nie okreslone przez Hoefer Inc do ochrony przewidzianej przez urzadzenie moze zostac obnizony Instrument ten jest przeznaczony do uzytku w laboratoriach kryty tylko Tylko akcesori w i cz ci zatwierdzone lub dostarczone przez Hoefer Inc mog by wykorzystane do eksploatacji utrzymania i obs ugi tego produktu korzysta jedynie zasilacza e jest nosz ce oznakowanie CE lub bezpiecze stwa uwierzytelnione przez uznane na poziomie krajowym laboratorium badawcze
12. fourni par Hoefer Inc pourrait tre utilis pour fonctionner maintenir et entretenir ce produit utilise Seulement une alimentation qui est CET a marqu ou la s curit certifi par un nationalement reconnu essayant le laboratoire Le couvercle de s curit doit tre sa place avant connecter l alimentation mene une alimentation Tourner tous contr les d alimentation de et d brancher les avances de pouvoir avant enlever le couvercle de s curit Circuler seulement de l eau ou 50 50 glycol d eau thyl ne par l exchanger de chaleur si si quip Ne pas connecter l exchanger de chaleur un robinet d eau ou la source d agent de refroidissement o la pression d eau est non r gul e Ne Jamais introduire d antigel ou du dissolvant organique dans n importe quelle partie de e piii l instrument Les dissolvants organiques causeront des dommages irr parables l unit Ne pas fonctionner avec les temp ratures de tampon au dessus du maximum a sp cifi des sp cifications techniques La surchauffe causera des dommages irr parables l unit Wichtige Informationen German Wenn diese Ausr stung gewisserma en nicht angegeben durch Hoefer Inc verwendet wird kann der durch die Ausr stung zur Verf gung gestellte Schutz verschlechtert werden Dieses Instrument wird f r den Innenlaborgebrauch nur daf r entworfen Nur Zus tze und Teile genehmigten oder lieferten durc
13. is warped replace along a straight path If the running tray is warped replace Cool agarose to 50 C to prevent the tray from warping Circulate buffer if it becomes depleted by stopping the run and pipetting the buffer from one chamber to the other Double banded pattern Make sure the comb remains vertical after the gel is cast so that the well shape is not distorted Decrease the buffer level to 1 mm above the top of the gel in order to reduce the temperature gradient in the gel Poor band resolution Add Ficoll glycerol or sucrose to the sample loading buffer to ensure that the sample sinks to the bottom of the well Ficoll is the recommended agent Make sure the sample is completely dissolved Reduce the sample concentration Reduce the sample volume Reduce voltage to 5 V cm Be sure the well floor is at least 1 mm thick to prevent samples from leaking through the bottom Reduce the salt concentration of the sample Check enzyme activity the sample may require longer digestion or a different restriction buffer Prepare fresh sample if you suspect nuclease contamination Choose agarose with a low endosmosis value Foam pads peel off Do not press the running tray into place Install as described on page 4 e p9 Important Do not adjust the pH of these buffers once they are prepared according to the recipe e plo Notes buffers and volumes
14. Running buffers for DNA in agarose gels Recipes for the three most commonly used running buffers for DNA electrophoresis are listed below The buffering capacity of both TBE and TPE is usually sufficient so that buffer circulation is unnecessary Circulation may be required during runs longer than 3 hours or when using the TAE buffer 1 10X Tris borate EDTA TBE stock buffer 0 89 M Tris 0 89 M boric acid 20 mM EDTA pH 8 3 1000 ml Tris base FW 121 1 0 89 M 108 0 g Boric acid FW 61 8 0 89 M 55 0g EDTA solution 0 02 M 40 0 ml 0 5 M pH 8 0 solution 4 Deionized H20 to 1000 0 ml Stir Do not adjust pH Before use dilute either to 0 5X to yield 45 mM Tris base 45 mM boric acid and 1 mM EDTA This dilution is often used because current remains low resulting in less heat 1X to yield 89 mM Tris base 89 mM boric acid and 2 mM EDTA 2 10X Tris phosphate EDTA TPE stock buffer 0 89 M Tris 0 89 M phosphoric acid 20 mM EDTA pH 8 1 1000 ml Tris base FW 121 1 0 89 M 108 0 g Phosphoric acid 85 0 23 M 15 5 ml EDTA solution 0 02 M 40 0 ml 0 5 M pH 8 0 solution 4 Deionized H20 to 1000 0 ml Stir Do not adjust pH Dilute to 1X to yield 89 mM Tris base 23 mM phosphoric acid and 2 mM EDTA 3 10X Tris acetate EDTA TAE stock buffer 0 4 M Tris 0 2 M acetic acid 10 mM EDTA pH 8 4 1000 ml Tris base FW 121 1 0 40 M 48 4 g Acetic acid 99 5 0 20 M 11 4 ml EDTA sol
15. glicol de agua etileno por el intercambiador de calor si se es el caso equiparon No conecte el intercambiador de calor a un toque de la agua ni cualquier fuente del l quido refrigerante donde la presi n del agua est libre Nunca introduce anticongelante ni alg n solvente org nico en cualquier parte del instrumento Los solventes org nicos causar n da o irreparable a la unidad No opera con temperaturas de b fer encima del m ximo especific especificaciones t cnicas Recalentar causar da o irreparable a la unidad Viktig Information Swedish om denna utrustning anv nds i ett s tt som inte har specificeras av Hoefer Inc skyddet tillhandah ll vid utrustningen kan skadas Detta instrument formges f r inomhuslaboratorium anv ndning bara Bara medhj lpare och delar godk nde eller levererade vid Hoefer Inc kan anv ndas f r fungera underh lla och servicing denna produkt anv nder bara en kraft tillg ng som r CE markerade eller s kerhet intygade vid en nationellt erk nd testande laboratorium S kerheten locket m ste vara p platsen f re koppla kraften tillg ngen blyen till en kraft tillg ng Vander sig alla kraft tillg ng kontroller av och kopplar bort kraften blyen f re flytta s kerheten locket Cirkulerar bara vatten eller 50 50 vatten ethylene glycol genom v rmen exchanger i s utrustad fall Inte kopplar v rmen exchanger till en vatten kran eller n got kylmedel k
16. h Hoefer Inc kann f r das Funktionieren das Aufrechterhalten und die Wartung dieses Produktes verwendet werden Verwenden Sie nur eine Energieversorgung die CE gekennzeichnet oder durch ein national anerkanntes Probelaboratorium bescheinigte Sicherheit ist Der Sicherheitsdeckel muss im Platz vor dem AnschlieBen der Energieversorgung sein f hrt zu einer Energieversorgung Alle Energieversorgungssteuerungen abdrehen und die Macht trennen f hrt vor dem Entfernen des Sicherheitsdeckels Nur Wasser oder 50 50 Glykol des Wassers Athylens durch den W rmeaustauscher wenn so ausgestattet in Umlauf setzen Verbinden Sie den W rmeaustauscher mit einem Wasserklaps oder jeder K hlmittel Quelle nicht wo der Wasserdruck ungeregelt wird F hren Sie nie Frostschutzmittel oder jedes organische L sungsmittel in jeden Teil des Instrumentes ein Organische L sungsmittel werden nicht wiedergutzumachenden Schaden der Einheit verursachen Mit Puffertemperaturen ber angegebenen technischen Spezifizierungen des Maximums nicht funktionieren Die berhitzung wird nicht wiedergutzumachenden Schaden der Einheit verursachen Informazioni Importanti Italian Se quest apparecchiatura usata in un modo specificato da Hoefer Inc la protezione fornito dall apparecchiatura potrebbe essere indebolita Questo strumento disegnato per l uso di laboratorio interno solo Solo gli accessori e le parti hanno approvat
17. indicated by the black dot and one at the center e p Final casting steps 0 Pour the agarose solution cooled to 50 C onto the running tray seated in the casting tray Orient the comb assembly so that it is at the end of the tray opposite the direction of migration typically at the cathode end which is marked by a black dot on the handle Fit the comb assembly into the slots o Allow a minimum of 30 min for the gel to set then remove the comb carefully partially lift and slightly tilt the comb at one end and slowly withdraw it from the gel Pulling the comb straight up creates a vacuum in the wells that may lift the gel out of the tray e Lift the running tray out of the casting tray and transfer it with the gel to the horizontal unit Orient the running platform so that the sample will run to red That is place the sample wells at the cathode gt end which is indicated by a black dot A notch on either side of the running tray centers the tray on the running platform Caution Wear UV safety goggles and protect skin when using a UV lamp Note Refer to the Buffers volumes and notes section for additional information and guidelines on page 10 See page 12 for a sample loading buffer recipe and well volumes for various comb sizes in gels of different thicknesses Important f running two sets of samples in one gel monitor the run closely and stop electrophoresis when the marke
18. n 0 5X TBE buffer at 150 V e p13 Note For an example of RNA electrophoresis refer to Molecular Cloning A Laboratory Manual by J Sambrook and D W Russell Caution Ethidium bromide is a known mutagen Always wear gloves when handling Caution Wear UV safety goggles and protect skin when using any UV light source Note Ethidium bromide slows DNA migration by 15 Note Minimize the staining time to prevent small nucleic acid fragments from diffusing out of the gel e pl4 RNA mobility RNA can also be separated on the basis of size To avoid irregularities due to secondary structure RNA is denatured either before or during electrophoresis For example RNA fragments previously denatured with glyoxal and dimethylsulfoxide can be separated on neutral agarose gels or RNA can be fractionated on agarose gels containing methylmercuric hydroxide or formaldehyde RNA samples usually require longer runs or buffers that are easily depleted and so require circulation The Hoefer SUB20C and SUB25C horizontal units are recommended for this appli cation rather than the HE33 DNA detection DNA can be detected either by the fluorescence of bound ethidium bromide or by autoradiogra phy of radio labeled DNA Ethidium bromide 0 5 pg ml can be added to running buffer to monitor sample progress because the dye s fluorescence reveals DNA under UV light To check band location turn off the power supply and remove
19. ncludes electrode posts 7 2x 14 2x 5 5 in Weight 0 82 kg 1 8 Ib base lid and leads only Product certifications EN61010 1 UL61010A 1 CSA C22 2 1010 1 CE Certified Before you start 1 Wash all components with a dilute solution of laboratory detergent and rinse thoroughly 2 Level the unit by placing the spirit level on the running platform and adjusting the leveling feet Volume for 3 mm thick gels tray size cm agarose ml 15 x 10 45 15x15 68 15 x 20 90 Caution Ethidium bromide is a known mutagen Always wear gloves when handling Operating instructions Agarose gels are first cast in the gel casting kit and samples are then loaded into the wells and electrophoretically separated The fluorescent dye ethidium bromide can be added to the gel or electrophoresis buffer or both in order to track separation progress At the completion of electrophoresis the gel may be stained and photographed blot transferred or dried for autoradiography Casting the gel Prepare the solutions 0 Prepare about 1 3 liters of running buffer Up to 100 ml of buffer is required for the gel and 1 2 liters for the buffer chamber Refer to page 10 for recipes of three commonly used electrophoretic running buffers o Prepare the sample loading buffer Refer to page 12 for a recipe and tabulated volume capacity for each comb size e Prepare agarose solution s Dissolve agarose in running b
20. o o hanno fornito da Hoefer Inc potrebbe essere usato per operare per mantenere e per revisionare questo prodotto usa Solo un alimentatore che CE ha marcato o la sicurezza certificato da un nazionalmente riconosciuto testando il laboratorio coperchio di sicurezza deve essere nel luogo prima di collegare i piombi di alimentatore a un alimentatore Spegne tutto i controlli di alimentatore e disinserisce i piombi di potere prima di togliere il coperchio di sicurezza Circola solo l acqua o 50 50 glicole di acqua etilene attraverso lo scambiatore di calore se cosi equipaggiato Non collegare lo scambiatore di calore a un rubinetto di acqua o qualunque fonte di refrigerante dove la pressione di acqua e sregolata Non introduce mai l antigelo o qualunque solvente organico in qualunque parte dello strumento solventi organici causeranno il danno irreparabile all unit Non opera con le temperature di tampone al di sopra del massimo ha specificato le descrizioni tecniche Il surriscaldamento causer il danno irreparabile all unit Viktig Informasjon Norwegian Hvis dette utstyret blir brukt i en m te ikke spesifisert ved Hoefer Inc beskyttelsen som ha blitt git av utstyret kan bli svekket Dette instrumentet er utformet for innend rs laboratoriumbruk bare Bare tilbehor og deler godkjente eller forsynte ved Hoefer Inc kan bli brukt for drive vedlikeholde og betjene dette produktet
21. of wells mm mm code number 1 2 1 5 113 10 HE91A P 1 5 1 2 3 0 113 10 HE91A P 3 0 10 1 5 9 7 HE91A 10 1 5 10 3 0 9 7 HE91A 10 3 0 15 1 0 7 1 HE91A 15 1 0 15 1 5 7 1 HE91A 15 1 5 15 3 0 7 1 HE91A 15 3 0 20 1 0 4 7 HE91A 20 1 0 20 1 5 4 7 HE91A 20 1 5 20 3 0 4 7 HE91A 20 3 0 30 1 0 3 0 HE91A 30 1 0 Preparative combs form two marker wells one on each side of the preparative well The first value in each column refers to the prep well the second to the reference well HE96X lid w power cables SE6056 HV high voltage leads set combs see list for sizes HE91 BK comb back with HE90X 2 screws buffer chamber HE99XRK 1 leveling foot e p17 Hoefer Inc 84 October Hill Road Holliston MA 01746 Toll Free 1 800 227 4750 Phone 1 508 893 8999 Fax 1 508 893 0176 E mail support hoeferinc com Web www hoeferinc com Hoefer is a registered trademark of Hoefer Inc 2012 Hoefer Inc All rights reserved Printed in the USA Hoefer
22. r dye approaches the wells in the center Preparing for electrophoresis 0 Optional To monitor separation progress either add 0 5 ug ml final conc of ethidium bromide to the running buffer now or add 50 ug ml final conc ethidium bromide to the sample buffer To visualize progress turn off the power supply remove the lid assembly and hold a portable UV lamp near the gel Note Adding ethidium bromide to the running or sample buffer slows migration slightly Detection by this method is not as sensitive as by staining after the electrophoresis run See the DNA detection section for more details page 14 e Fill the chamber with buffer until the gel is submerged z1 mm e Load the samples Add the sample to 1 5 volume of the sample loading buffer Mix each sample and load into a well with a micro pipet taking care to avoid puncturing the well bottom or entrapping bubbles o Place the lid on the unit so that the cathode black lead is at the end nearest the samples Nucleic acid samples migrate toward the anode e Connect the color coded leads red to red and black to black to an approved power supply Set the voltage and timer if available Agarose gels are typically run at constant voltage under a voltage gradient in the range of 2 5 V cm The distance between the electrodes is z26 cm so a setting of 130V results in a gradient of 5 V cm e ps After electrophoresis o Importan
23. rolos de estoque de poder e desconecta os chumbos de poder antes de retirar a tampa de seguran a Circulam s gua ou 50 50 glicol de gua ethylene pelo exchanger de calor se for assim equiparam N o ligue o exchanger de calor a uma torneira de gua nem qualquer fonte de refrigerante onde a press o de gua n o regulado Nunca introduz anticongelante nem qualquer org nico solvente em qualquer parte do instrumento Org nico solvente causar agress o irrepar vel unidade N o opera com temperaturas de buffer acima do m ximo especificou especificac es t cnicas Superaquecer causar agress o irrepar vel unidade e pv Informaci n Importante Spanish Si este equipo es utilizado en una manera no especificado por Hoefer Inc la protecci n proporcionado por el equipo puede ser da ada Este instrumento es dise ado para el uso interior del laboratorio s lo S lo accesorios y partes aprobaron o suministraron por Hoefer Inc puede ser utilizado para operar para mantener y para atender a este producto S lo utiliza una alimentaci n que es CE marc o la seguridad certificada por un nacionalmente reconocido probando el laboratorio Latapa de la seguridad debe estar en el lugar antes de conectar la alimentaci n lleva a una alimentaci n Apaga todos controles de alimentaci n y desconecta los plomos del poder antes de quitar la tapa de la seguridad Circula s lo agua o 50 50
24. t Always turn off the power supply and disconnect the leads before removing the lid o If no ethidium bromide was added to the gel or sample before the run stain the gel now in a solution of 0 5 to 1 0 ug ml ethidium bromide in water or buffer e Clean the unit as described in the next section Care and maintenance Cleaning Never autoclave or heat any component above 45 C Never use abrasive cleansers Do not expose the unit to solutions or vapors of aromatic or halogenated hydrocarbons ketones esters alcohols over 3096 or concentrated acids over 25 The unit is resistant to all common electrophore sis buffers but we recommend a thorough wash ing with a mild detergent after each use Rinse with distilled water and allow to air dry To remove DNase and RNase contamination fill the unit with 3 hydrogen peroxide H202 soak for 10 minutes then rinse thoroughly with DEPC treated autoclaved deionized water Sambrook and Russell et al 1 7 82 Troubleshooting problem solution Sample well deformed Allow the gel to set for a minimum of 1 hour and make sure it is at room temperature before removing the comb Remove the comb at a slight angle and very slowly to prevent the gel from breaking Take care to not damage the well with the pipet while loading the sample aim for the center of the well and do not puncture the bottom with the pipet tip Samples not running If the comb
25. taa olla avuton Tama valine suunnitellaan sis laboratoriok yt lle vain Vain lis varusteet ja osat hyv ksyiv t tai toimitti Hoefer Inc oheen voi k ytt k ytt miselle valvoalle ja servicing t m tuote Vain k ytt k ytt j nnitett joka on CE merkitsi tai turvallisuus joka on todistanut aidoksi ohi joka on kansallisesti tunnustettnut testaaminen laboratoriota Turvallisuuskansi t ytyy olla paikallaan k ytt j nnitteeseen Kiert kaikki kaytt jannitevalvonnat ja irrottaa valtalyijyt ennen poistaminen turvallisuuskantta Kiert vain vesi tai 50 50 vesi ethylene glycol siin tapauksessa varustetun limm nvaihtimen l pi l yhdist l mm nvaihdinta vesinapautukseen eik j hdytysnestel hteeseen miss vesipaine on unregulated Pakkasneste eik orgaaninen liuotin v lineen osassa ei esitele Koskaan Orgaaniset liuottimet aiheuttavat korvaamattoman vahingon yksikk n Ei k yt puskuria yll olevia l mp tiloja enint n m ritetyill teknisill t smennyksill Ylikuumeneminen aiheuttaa korvaamattoman vahingon yksikk n Information Importante French Si cet quipement est utilis dans une mani re pas sp cifi par Hoefer Inc la protection fourni par l quipement pourrait tre diminu e Cet instrument est con u pour l usage de laboratoire int rieur seulement Seulement les accessoires et les parties ont approuv ou ont
26. the lid of the agarose unit Hold a portable UV lamp near the running tray Replace the lid and turn on the power again to resume electrophoresis Alternatively after electrophoresis stain the gel in an ethidium bromide solution 0 5 pg ml H20 for 15 to 60 minutes and then view or photograph the sample on a UV transilluminator To photograph the gel either place the running tray on the transilluminator surface or slide the gel onto the surface for maximum exposure The running tray is 95 transparent to 302 nm light and 40 transparent to 254 nm light If you place the gel on the transilluminator ensure that it lies flat by cutting off the ridges formed by the grooves in the running tray Do not damage the transilluminator surface trim both ends of the gel with a spatula while it is still in the tray lift away the ridges and then slide the gel onto the transilluminator For viewing 302 nm light is recommended for both acceptable sensitivity and reduced photonicking To reduce the background fluorescence of unbound ethidium bromide the gel can be destained by soaking it for 5 minutes in 0 01 M MgCh or for 1 hour in 0 001 M MgSO4 Destaining makes it easier to detect small quan tities less than 10 ng of DNA Sambrook and Russell A9 4 Transfer Before transfer trim off the ridges at both ends of the gel to ensure even gel contact with the membrane Bibliography Ausubel et al eds Current Protocols in
27. uffer heat according to instructions accompanying the agarose and allow the solution to cool to 50 C before pouring into the running tray Optional Add 0 5 ug ml ethidium bromide to the gel solution in order to facilitate observation of separation progress during electrophoresis Fig 2 Gel casting kit Running tray installation Approach the foam pad with one end of the running tray Arrow A and then gently press the tray edge against the pad compressing it enough to allow the opposite end of the running tray to drop fully into the casting tray Arrow B before sealing against the foam pad Note Grooves in the running tray create ridges at both ends of the gel to prevent it from slipping or floating If these ridges are not desired either tape over the grooves before casting or trim off ridges with a spatula after the run e p4 Prepare the casting tray and pour the gel 0 Install a foam pad at each end of the casting tray Use a comb as a placement guide so that the pad adheres 1 mm from the bottom of the tray Lay the comb into the bottom of the tray oriented so that it fits completely across the tray along the side that is 16cm wide Peel off the adhesive backing on the foam pad align the pad on the comb adhesive side toward the inside wall of the tray and slide the comb against the wall Press the foam pad in place and repeat with second pad on the wall opposite the first pad Color coded dots
28. ution 0 5 M pH 8 0 solution 4 0 01 M 20 0 ml Deionized H20 to 1000 0 ml Stir Do not adjust pH Dilute to 1X before use to yield 40 mM Tris base 20 mM acetic acid and 1 mM EDTA 4 EDTA solution ethylenediamine tetraacetic acid 0 5 M pH 8 0 100 ml Na2EDTA 2H20 FW 372 2 0 5 M 18 6g Deionized H20 to 70 0 ml NaOH 10 M to pH 8 0 5 0 ml Deionized H20 to 100 0 ml Sambrook J and Russell D W 2001 Molecular Cloning A Labora tory Manual A1 17 Current Protocols in Molecular Biology 1993 A 2 1 e pll e p12 Loading buffer and sample volumes Loading buffer 5X 25 Ficoll 400 0 25 Bromphenol blue 10 ml Deionized H20 to 7 0 ml Ficoll 400 258 Bromophenol blue FW 691 9 25 0 mg Deionized H20 to 10 0 ml Add 1 volume loading buffer to 4 volumes of sample Loading buffer increases solution density Note 1 Sucrose or glycerol may be used instead of Ficoll 400 Note 2 Xylene cyanol 0 25 which migrates more slowly than bromophenol blue can be added as an additional marker if desired The agarose concentration determines the position of the dye bands relative to a polynucleotide Tracking dyes may be omitted to eliminate obscuring or dragging effects caused by comigration with smaller nucleic acids Comb specifications and well volumes well well sample vol code no of width thickness per 1 mm number wells mm mm depth ul HE91A P 1 5 1 2 113 10 15 171 14 5
29. ver introduce antifreeze or any organic solvent into any part of the instrument Organic solvents will cause irreparable damage to the unit Do not operate with buffer temperatures above the maximum specified technical specifications Overheating will cause irreparable damage to the unit Dule it Informace Czech Pokud by toto za zen je pou ito zp sobem kter nen podle Hoefer Inc ochrana poskytovan na z klad za zen m e b t naru ena Tento n stroj je ur en pro vnit n pou it v laborato i pouze Pouze p slu enstv a sti schv len nebo poskytnut ch Hoefer Inc mohou b t pou ity pro provoz dr bu a dr b tohoto v robku zdroj nap jen pou vaj jen e je opat en ozna en m CE osv d ena nebo bezpe nost vnitrost tn uznan mi zku ebn mi laborato Bezpe nosti lid mus b t zavedena p ed p ipojen m nap jec zdroj nap jen vede k Turn ve ker nap jen kontroly vypnuto a odpojit p ed odb rem energie vede bezpe nostn v ko Rozeslat pouze voda nebo 50 50 voda ethylenglykolu prost ednictv m v m n k tepla je li to vybavena Nemaj p ipojen v m n k tepla s vodn mi set epn nebo jak koli chladic kapaliny zdroje kde tlak vody je neregulo Nikdy zav st prost edek proti zamrznut nebo jak koli organick rozpou t dla do jak koli sti z tohoto n stroje Rozpustidl m zp sob nenapraviteln po kozen jednotka
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