Hoefer HB1000
Contents
1. Hybridize at lower salt higher temperature Wash with lower salt higher temperature Incubate with very low salt change nuclease solution Use a smaller probe or a different probe Clean probe of contaminants prior to use 4 Agueous or denaturing hybridization buffer If hybridization takes place in an agueous salt environment of 0 8 to 1 2 M salt the Tm the temperature at which half of the duplex molecules will dissociate under a given set of conditions can be 90 C This is high enough to degrade DNA RNA and some proteins It may be necessary to add formamide as a denaturing temperature lowering agent For every percent of formamide added to the reaction the Tm is reduced by 0 65 C Therefore at 80 formamide reactions can be performed in the 40 55 C range On the other hand the presence of formamide reguires longer incubations since the rate of formamide based hybridization is at least three fold lower than that of agueous hybridization Protocol D Random priming method for tagging DNA with fluorescein labeled nucleotide or other labeled nucleotides This method uses DNA polymerase to incorporate fluorescein 11 dUTP into double stranded DNA probes This protocol can also be used to incorporate any tagged nucleotides Eguipment e Micropipettes and tips e Microcentrifuge e 1 5 ml microcentrifuge tubes e Cap lock for microcentrifuge tube e Boiling water bath e Water bath set to 37 C2. o p15 o pl6 Reagents e Deionized sterile water e EDTA 0 5 M e DNA polymerase Klenow fragment 4 5 units uL e Nucleotide mix 300 uM each of dATP dCTP dGTP and 60 uM dTTP e Random nonamer 9 mer primers 2 5 ug uL in water e Reaction buffer 10X 50 mM MgClo 10 mM 2 mercaptoethanol 500 mM Tris HCI pH 7 5 e Tagged nucleotide fluorescein 11 dUTP e Template DNA in water 5 ng mL Procedure 1 Pipette 10 uL of template DNA plus 10 uL of water into a microcentrifuge tube and cap tightly Secure cap with a cap lock or use a bent paper clip to ensure that the cap does not pop when the contents are boiled 2 Place the tube into the boiling water bath for 5 minutes 3 Immediately place tube on ice for 5 minutes 4 Centrifuge for 15 seconds in microcentrifuge 5 Add the reagents listed below to a fresh tube on ice in the following order a 10 uL Nucleotide mix b 5 ul Tagged nucleotide c 5 yl Reaction buffer 10X d 5 ul Random primers e 10 ul Boiled DNA f 14 ul Water g 1 uL DNA polymerase Klenow fragment 6 Mix gently and incubate at 37 C for 1 hour 7 Stop the reaction by adding 2 ul EDTA 8 Store probes at 20 C in the dark Note When preparing prehybridization hybridization solutions add dry reagents directly to the formamide SSC solution Incubate with mixing at 40 50 C for 2 hours or until dissolved Store at 20 C SDS will precipitate at room temperature
3. manual Hybridization Oven um HB1000 IM Rev D0 12 12 Hoefer Page finder important KTO E d e EE L Waste Electrical and Electronic Equipment WEEE vill MELOU CEO NE bd ke a A 1 SOS O MIC ALTON eege 3 Setup INSTFUC TROTS aven m ne h je u o NRA ERE Arr 4 Operating INSTITUCION SS cuasi ia ud ake 6 Care and ut ht ie 10 Customer service Information 10 Hybridization Techngues orons 11 Experimental porotocols eee 13 Ke 20 Ordering KUER eeben vaasa mass 21 Important Information English If this eguipment is used in a manner not speci fied by Hoefer Inc the protection provided by the eguipment 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 main taining and servicing this product Warning Because this instrument can develop sufficient voltage and current to produce a lethal shock care must be exercised in its operation This instrument is designed in accordance with the EN61010 1 2001 electrical safety standard Nevertheless it should be used only by properly trained operators Read this entire manual before using the instrument and use only according to the instructions The instrument must always be used with the earth lead of the power cord correctly grounded to earth at the mains outlet Use only undamaged electrical wire and eguip ment spec
4. detection reagent 2 e Membrane following hybridization Procedure 1 Mix equal volumes of detection reagents 1 and 2 2 Pipette the mixture over the surface of the membrane and leave at room temperature for 1 minute 3 Follow the wash instructions provided by the kit manufacturer Remove the final wash and cover the membrane in plastic wrap 4 Image using the Hoefer ULTima Pro chemilumines cence imaging system References Ross J 1998 Nucleic Acid Hybridization Essential Technigues John Wiley and Sons ISBN 0 471 97125 1 Sambrook J 2001 Molecular Cloning A Laboratory Manual Third Edition Cold Spring Harbor Labora tory Press ISBN 0 87969 577 3 Current Protocols in Molecular Biology 1987 John Wiley and Sons ISBN 0 471 50338 X Ordering information product guantity code number HB1000 Hybridization Oven 1 HB1000 115V 115 VAC 60 Hz HB1000 Hybridization Oven 1 HB1000 230V 230 VAC 50 Hz Bottle small 35 x 150 mm 1 HB1110X Includes Borosilicate glass bottles with polypropylene cap and Teflon seal Bottle large 35 x 300 mm 1 HB1111X Includes Borosilicate glass bottles with polypropylene cap and Teflon seal Rocker plate HB1140X Drip trays HB1141X Carousel kit for small or large bottles HB1109X Adjustable feet HB1150X Power cord 115 V Power cord 230 V Euro Plug Schuko Fuses 10 amp 250 V 3AG SB Cer PSCORD 115V PSCORD 230V PSF10A SB 3AG O FE E ER IN gt o p21
5. excess Fig 6 right N N pun pu O Q gt gt x Q Q 2 2 o a Amount of target nucleic Amount of target nucleic acid arbitrary units acid arbitrary units o p13 o p14 2 Length of probe The goal is to increase hybridization efficiency while minimizing background The larger the probe the more efficiently it binds target The smaller the probe however an ever greater number of probe molecules is present per unit amount ng In most cases probes range from 20 1000 bps 3 Salt concentration and temperature Nucleic acid hybridization requires both salt monova lent cations such as Na to counter the charge effects of the phosphate backbone and heat to provide non denaturing kinetic energy that drives the slow nucleation step Because the effects of each variable affect the other knowing the thermostability of the hybrid probe target is helpful The hybridization rate varies directly with the sodium ion concentration between 0 03 and 1 2 M Most protocols call for an environment between 0 5 and 1 1 M Nat Situation G C 45 55 G C lt 45 G C gt 55 Evidence of probe target mismatching Target and probe are degraded in an agueous environment Unacceptable high background Response Follow normal protocol Lower salt and temperature Raise salt and temperature Lower salt and temperature Hybridize in a formamide based buffer solution Use less probe
6. rios e partes aprovaram ou forneceu por Hoefer Inc pode ser usada para operar manter e servicing este produto Advertindo Porque este instrumento pode desen volver voltagem suficiente e corrente produzir um choque letal cuidado deve ser exercitado em sua opera o Este instrumento projectado de acordo com o EN61010 1 2001 condi o de seguran a el ctrica N o obstante deve ser usado s por operadores adequadamente treinados Leia este manual inteiro antes de usar o instrumento e use s de acordo com as instrucc es O instrumento sempre deve ser usado com o chumbo de terra do cord o de poder corretamente baseou a terra nos canos saida principais Usa fio el ctrico s intacto e eguipamento espec fico para as voltagens que voc usar Todo egui pamento conectado a voltagem alta deve ser de acordo com EN61010 1 2001 Mantem o instrumento t o seco e limpo como poss vel Limpe regularmente com um pano h mido macio Deixe o instrumento secar comple tamente antes de uso N o opera o instrumento em humidade extrema acima de 80 Evite condensac o deixando o eguilibrate de unidade a temperatura ambiental guando tomar o instrumento de um mais frio a um ambiente mais guente Permitir esfriar suficiente assegura gue as abertu ras do instrumento n o s o cobertas Informaci n Importante Spanish Si este eguipo es utilizado en una manera no espe cificado por Hoefer S a la protecci n proporci
7. sont pas couverts Wichtige Informationen German e 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 Innenlaborge brauch nur daf r entworfen Nur Zus tze und Teile genehmigten oder lieferten durch Hoefer Inc kann f r das Funktionieren das Aufrechterhalten und die Wartung dieses Produk tes verwendet werden Die Warnung Weil dieses Instrument gen gend Stromspannung und Strom entwickeln kann um einen t dlichen Sto zu erzeugen muss Sorge in seiner Operation ausge bt werden Dieses Instrument wird in bereinstimmung mit dem EN61010 1 2001 elektrischen Sicherheitsstan dard daf r entworfen Dennoch sollte es nur von richtig erzogenen Maschinenbedienern verwendet werden Lesen Sie dieses komplette Handbuch vor dem Verwenden des Instrumentes und verwenden Sie nur gem den Instruktionen Das Instrument muss immer mit der Erdleitung der Macht Schnur richtig niedergelegt zur Erde am Hauptausgang verwendet werden Nur unbesch digte elektrische Leitung und Ausris tung spezifisch f r die Stromspannungen verwen den die Sie verwenden werden Die ganze mit der Hochspannung verbundene Ausr stung sollte in Ubereinstimmung mit EN61010 1 2001 sein Das Instrument ebenso trocken halten und reini gen wie m glich Wischen Sie regelm ig mit einem weichen b
8. 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 ECL is a trademark of GE Healthcare formerly Amersham Biosciences O 2012 Hoefer Inc All rights reserved Printed in the USA
9. but remain in solution at 37 C Protocol Hybridization to Nylon or Nitrocellulose Hybridization of probe to target nucleic acid that has been previously transferred to nylon or nitrocel lulose membranes is accomplished by adding single stranded probe to the membranes that have first been incubated with prehybridization solution Both the prehybridization and hybridization solutions contain buffers designed to prevent adventitious binding of the probe to the filters Reagents and eguipment e Prehybridization hybridization solution 45 formamide 5X SSPE 0 9 M NaCl 50 mM sodium phosphate buffer pH 7 4 5 mM EDTA 0 1 SDS 5X Denhardt s solution 0 1 each of Ficoll poly vinylpyrrolidone and bovine serum albumin and 100 mg mL of denatured salmon sperm DNA Mix well and remove aggregates before use e Hoefer hybridization bottle s and caps e 15 mL plastic tube e Boiling water bath e Bucket of ice e Gloves e Beta blocking shield e Hoefer HB1000 Hybridization Oven e p17 o p18 Procedure 1 Add 15 mL of prehybridization solution to each hybridization bottle containing the blot Remove bubbles between the glass and blot Cap the bottles attach the bottles to the wheels of the bottle holder carousel and close the hybridization oven door 2 Incubate the blot at 42 C for 1 hour 3 Remove the prehybridization solution and replace with 10 mL of hybridization solution 4 If using radiolabele
10. ciente asegure que las aberturas del Viktig Information Swedish e om denna utrustning anv nds i ett s tt som inte har specificeras av Hoefer Inc skyddet tillhan dah ll vid utrustningen kan skadas e Detta instrument formges f r inomhuslaborato rium anv ndning bara Bara medhj lpare och delar godk nde eller lever erade vid Hoefer Inc kan anv ndas f r fungera underh lla och servicing denna produkt e varna D rf r att detta instrument kan utveckla tillr cklig sp nning och str m att producera en d dlig st t m ste vas omsorg i dess funktion e Detta instrument formges i verensst mmelse med EN61010 1 2001 elektriska s kerheten stan darden Icke desto mindre b r det anv ndas bara av riktigt utbildade operat rer L s denna hela handbok f re anv nda instrumentet och anv nd bara enligt undervisningarna e Instrumentet m ste alltid anv ndas med jorden blyet av kraften repet riktigt grounded till jorden p det huvudutloppet e Anv nder bara undamaged elektrisk tr d och utrustning specifik f r sp nningarna du ska anv nda All utrustning kopplats som till h g sp n ning skulle vara i verensst mmelse med EN61010 1 2001 H ller instrumentet d torkar och reng r som m jlig Torka regelbundet med en mjuk fuktig trasa L t instrumentet torka fullst ndigt f re anv ndningen Fungerar inte instrumentet i extrem fuktighet ver 80 Undvik kondensering vid l ta enheten
11. d probes work safely from the other side of a beta blocking shield Pipette 1 x 10 counts per minute of radiolabeled probe or 200 ng of biotinylated probe into a 15 ml plastic tube Seal the tube with a plastic cap and poke a hole in the top with a syringe needle to prevent pressure build up during boiling 5 Denature the probe by placing the samples in the boiling water bath and heating for 10 minutes Immediately transfer the tube to ice for 5 minutes to prevent renaturation Add 5 mL of hybridiza tion buffer to the probe and transfer to the hybrid ization bottle containing the blot AVOID pouring the probe directly onto the blot 6 Incubate in the Hoefer HB1000 Hybridization Oven for 6 to 8 hours at 42 to 56 C Washing the blot e Tupperware container sized to contain the blot e 0 1X SSC 0 1 SDS pre warmed to 50 C e 2X SSC 0 1 SDS room temperature e 2X SSC room temperature e 0 15X SSC 0 1 SDS pre warmed to 50 C e Gloves e Filter paper e Cardboard e Plastic wrap e Tape For radioactive probes you will also need e X ray film holder e X ray film e Intensifying screen Non radioactive probes 1 Wash blots in 2x SSC 0 1 SDS for 3 minutes at room temperature repeat one 2 Wash filter in 0 15X SSC 0 1 SDS for 15 minutes at 50 C repeat once 3 Store blots in 2X SSC at room temperature Radioactive probes 1 Remove blot from hybridization bottle and transfer to Tupperwa
12. dnie z tym EN61010 1 2001 Bezpiecze stwo elektryc zne standard Niemniej jednak nale y stosowa jedynie przez odpowiednio przeszkoleni opera tor w Znajd pa stwo to ca y podr cznika przed zastosowaniem instrumentu i stosowa jedynie zgodnie z instrukcjami Instrument musi zawsze by wykorzystane z ziemi doprowadzi do zasilania detonuj cego w a ciwie uzasadnione na ziemi w sieci wodoci gowej pv rynku zbytu Wykorzystanie tylko nieuszkodzona elektrycznych drut w i urzadzenia specjalne do napie zaptaca wykorzystania Wszystkie urzadzenia podtaczone do wysokiego napiecia powinny by zgodne z EN61010 1 2001 Kontrolowac instrumentu jako suche i czyste jak to mo liwe Wytrze regularnie przy pomocy miekkiego wilgotnej szmatki Niech sie instru mentem ca kowicie wysuszy przed u yciem Nie prowadz do instrumentu w skrajnych wilgotno ci powy ej 80 Zapobiec kondensacji najmu przez jednostk r wnowa y do temper atury pokojowej przy podejmowaniu instrumentu z ch odniejsze w cieplejszych rodowiska Aby umo liwi wystarczaj ce ch odzenia zapewniaj e rozci cia of the instrument nie obj te ubezpieczeniem Informa es Importantes Portuguese Se este equipamento usado numa maneira n o especificada por Hoefer Inc que a protec o forne cida pelo equipamento pode ser comprometida Este instrumento projectado para uso de interior de laborat rio s S acess
13. ee bases of one strand comple ments to those on the second strand If the remainder of the strands are complementary the two strands will anneal or zipper together very guickly Fig 5 The rate limiting step in nucleic acid hybridization is the duplex forma tion which again explains why probe to target concentrations are critical Step 1 Nucleation slow AO A6 Step 2 Zippering fast LS a SD j OI gt ua DEST gt TM TTT TTT 5 er Cake Fig 6 Analysis for probe excess in hybridization reactions Experimental protocols There are many different protocols available on the web in journals and in textbooks We refer ence several at the end of this section 1 Concentration of species Target The ideal guantity of target molecule depends on the complexity of the target nucleic acid The amplified DNA for example may have been expressed in large or small amounts Therefore the trick is to start with sufficient target 25 ug and determine an ideal amount experimentally Probe Plan to have more probe than expected target To determine this perform an extinction experiment serially increase the amount of target by 2 fold against a fixed amount of probe Hybridize for a short length of time and guantitate the amount of probe that has hybridized As long as the signal increases and shows linearity there is excess probe present Fig 6 left If the signal levels off then the probe is not present in
14. efeuchten Sie Stoff Lassen Sie das Instrument trocken v llig vor dem Gebrauch Das Instrument in der ufsersten Feuchtigkeit uber 80 nicht bedienen Vermeiden Sie Kondensa tion die Einheit eguilibrate zur Umgebungstem peratur laBend wenn Sie das Instrument von einem k lteren bis eine w rmere Umgebung nehmen Um das gen gend Abk hlen zu erlauben stellen Sie sicher dass die ffnungen des Instrumentes nicht bedeckt werden Informazioni Importanti Italiano Se guest apparecchiatura amp usata in un modo specificato da Hoefer Inc la protezione fornito dall apparecchiatura potrebbe essere indebolita Questo strumento disegnato per l uso di labora torio interno solo Solo gli accessori e le parti hanno approvato o piv hanno fornito da Hoefer Inc potrebbe essere usato per operare per mantenere e per revisionare guesto prodotto Avvertendo Perch guesto strumento pu sviluppare il voltaggio sufficiente e la corrente di produrre una scossa letale la cura deve essere esercitata nella sua operazione Ouesto strumento amp disegnato conformemente all EN61010 1 2001 la norma di sicurezza elettrica Tuttavia dovrebbe essere usato degli operatori solo correttamente addestrati Leggere guesto manuale intero prima di usare lo strumento e l uso solo secondo le istruzioni Lo strumento deve essere sempre usato col piombo di terra della spina di alimentazione corret tamente hanno messo a terra alla te
15. equilibrate till omgivande temperatur n r ta instru mentet fr n en kallare till en varmare milj Att till ta tillr cklig kyla ser till att h len av instru mentet inte t cks pvii O pviii English X French amp German X EES Italian amp az Spanish 156 Swedish Y Waste Electrical and Electronic Eguipment WEEE This symbol indicates that the waste of electrical and electronic eguipment 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 eguipment Ce symbole indique que les d chets relatifs l quipement lectrique et lectronigue 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 tes zu erhalten Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti come r
16. es et les parties ont approuv ou ont fourni par Hoefer Inc pourrait tre utilis pour fonctionner maintenir et entrete nir ce produit Avertissant Parce que cet instrument peut d vel opper la tension et le courant suffisants pour produire un choc mortel le soin doit tre exerc dans son op ration e Cet instrument est conform ment con u a VEN61010 1 2001 norme de s curit lectrique N anmoins il devrait tre seulement utilis par les op rateurs convenablement entra n s Lire ce manuel entier avant utiliser l instrument et l usage seulement selon les instructions e l instrument toujours doit tre utilis avec l avance de terre du cordon d alimentation correctement a fond a la terre a la sortie principale Utiliser le fil et l quipement lectriques seule ment intacts sp cifiques pour les tensions que vous utiliserez Tout quipement connect a haute tension devrait tre conform ment a EN61010 12001 e Garder instrument aussi sec et propre comme possible Essuyer r guli rement avec un doux touffer du tissu Laisser instrument seche compl tement avant l usage Ne pas fonctionner l instrument dans l extr me humidit au dessus de 80 Eviter la condensa tion en laissant l equilibrate d unit la temp ra ture ambiante en prenant l instrument d un plus froid un environnement plus chaud e Permettre le refroidissement suffisant garantir que les conduits de l instrument ne
17. he event of an accidental breakage e Never overtighten caps on bottles Hand tightening is sufficient e f the cap is difficult to unscrew NEVER ATTEMPT to force the bottle cap open Allow the bottle to cool and retry If the cap remains stuck discard the bottle e Bottles should not be used at temperatures above 70 C Caution Always unplug unit from electrical supply before cleaning or drying the unit IMPORTANT Request a copy of the Hoefer Inc Health and Safety Declaration form before returning the item No items can be accepted for servicing or return unless this form is properly completed e p10 Care and maintenance Cleaning The units are built to provide you with trouble free operation To ensure correct operation e Wipe ANY water from inside and outside the unit with a soft cloth or sponge e Use soap and water with a soft cloth or sponge to clean the unit e Do not allow chemicals to remain on unit surfaces e Never clean unit with abrasive pads or cleaners e Never clean unit with acetone or chloroform Customer service information Technical service and repair Hoefer Inc offers complete technical support for all of our products If you have any ques tions about how to use this product or would like to arrange to repair it please call or fax your local Hoefer Inc representative Check the Hoefer Inc website at www hoeferinc com for the distributor in your area Or contac
18. iettemin zou het enkel door goed getrainde bedieningslieden moeten worden gebruikt Lees dit volledige handboek voor het gebruik het instrument en gebruik enkel volgens de instructies Het instrument moet altijd met de aardeleiding van het stroomsnoer correct grondde naar aarde aan het hoofdafzetgebied worden gebruikt Gebruik enkel onbeschadigde elektrische draad en uitrustings specifiek voor de spanningen u zult gebruiken Alle uitrustingen sloten aan aan hoog spanning zou in overeenstemming met EN61010 1 2001 moeten Zijn Houd het instrument zo droge en schone zoals mogelijk Bij Wis regelmatig met een zacht temper doek Verhuur het instrument droogt volledig voor het gebruik Bedien niet het instrument in extreme vochtigheid bovenstaande 80 Vermijd condensatie door het verhuren van de eenheid in evenwicht brengt naar omgevingstemperatuur wanneer nemen het instrument van een kouder naar een lievere omgeving Om toe te staan voldoende afkoelen verzeker dat de luchtopeningen van het instrument niet bedekt zijn T rke Tietoa Finnish Jos t t varusteita k ytet n tavassa ei m ritetty Hoeferille Inc suojelu ehk isty varusteille saattaa olla avuton T m v line suunnitellaan sis laboratoriok yt lle vain Vain lis varusteet ja osat hyv ksyiv t tai toimitti Hoeferin oheen Inc voi k ytt k ytt miselle valvoalle ja servicing t m tuote Varoittaminen Koska t m v line
19. ific for the voltages you will use All eguipment connected to high voltage should be in accordance with EN61010 1 2001 Keep the instrument as dry and clean as possible Wipe regularly with a soft damp cloth Let the instrument dry completely before use Do not operate the instrument in extreme humid ity above 80 Avoid condensation by letting the unit eguilibrate to ambient temperature when taking the instrument from a colder to a warmer environment To permit sufficient cooling ensure that the vents of the instrument are not covered Dule it Informace Czech Pokud by toto za zen je pou ito zp sobem kter nen podle Hoefer ochrana poskytovan na z klad Inc 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 poskyt nut ch Hoefer Inc mohou b t pou ity pro provoz dr bu a dr b tohoto v robku Pozor Proto e tento n stroj m e vyvinout dostate n nap t a proud kter m vyr b t a smrtiacej ok p e mus b t vykon v na v jeho provoz Tento n stroj je ur en v souladu s EN61010 1 2001 elektrick bezpe nostn normy P esto e by m ly b t pou ity pouze dn vy kolen oper tor st cel toto ru n p ed pou it m n stroje a pou it pouze v souladu s pokyny P stroj mus b t v dy pou vaj se na v konu zemi vv o v st ra
20. ifiuti 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 simbolo 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 IMPORTANT Please read these instructions before operating your Hybridization Oven to familiarize yourself with operation procedures Introduction The Hoefer HB1000 Hybridization Oven is a cost effective high pertormance 10 bottle hybridization oven with state of the art micro processor temperature and variable speed controls The HB1000 Hybridization Oven can be set to operate anywhere from 10 to 18 RPM enabling consistent saturation of samples whether it be for washing or hybridizing Having the ability to remove the bottle holder carousel and replace it with an interchangeable rocker plate enables all hybridization protocols in one reliable benchtop unit An easily removable protective tray
21. ken f r bruking instrumentet og bruken bare gi til instruksjonene Instrumentet m alltid bli brukt med jorden blyet av kraftkabelen som riktig ha blitt jordet til jord p hovedledningen utl p Bruker bare uskadd elektrisk ledningsfremf ring og utstyr som er spesifikk for spenningene du vil bruke All utstyr koplet til h yspenning burde v re i samsvar med EN61010 1 2001 Beholder instrumentet som t rker og rengj r som mulig Visk regul rt med et mykt fuktig stoff La instrumentet t rker komplett f r bruk Driver instrumentet i ekstrem fuktighet ikke oven for 80 Unng kondensasjon ved la enheten equilibrate til omgivelsestemperatur ved taen instrumentets fra et kaldere til et varmere milj Til tillate tilstrekkelig kj lig sikrer at ventilasjon s pningene av instrumentet er ikke dekket Wazne Informacje Polish Je eli ten sprzet jest wykorzystywany w spos b nie okreslone przez Hoefer Inc do ochrony przewid zianej przez urzadzenie mo e zostac obnizony Instrument ten jest przeznaczony do u ytku w laboratoriach kryty tylko Tylko akcesori w i czesci zatwierdzone lub dostarc zone przez Hoefer Inc moga by wykorzystane do eksploatacji utrzymania i obstugi tego produktu Uwaga Poniewa ten akt prawny mo e by rozwini cie odpowiednich napi cie i bie cych do wyprodukowania miertelnego szoku opiek musi by wykonywane w dzia aniu Ten instrument zosta zaprojektowany zgo
22. l instruktionerne Instrumentet skal altid bruges med jordblyet af netledningen rigtigt jordede til jord p hovedled ningsudl bet O pii Bruger bare uskadt elektrisk tr d og udstyr som vaere specifik for spaendingerne du vil bruge Alt udstyr forbundet til hoj spaending skulle vaere i overensstemmelse med EN61010 1 2001 Beholder instrumentet s t r og ren som mulig T r regul rt med et bl dt fugtigt stof Lad instru mentt rken komplet f r brug Driver ikke instrumentet i yderst fugtighed oven for 80 Undg kondensation ved lade enheden equilibrate til omgivende temperatur ved tageen instrumentets fra et koldere til et varmere milj At tillade tilstr kkelig afk ling forsikrer at lufthullerne af instrumentet er ikke d kket Belangrijke Informatie Dutch Indien deze uitrusting in een manier wordt gebruikt die niet door Hoefer is gespecificeerd Nv 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 Nv kan voor het bedienen worden gebruikt handhavend en onderhouden van dit product Waarschuwend Omdat dit instrument voldoende spanning en stroom kan ontwikkelen om een dodelijke schok te produceren moet zorg in zijn operatie worden geoefend Dit instrument is in overeenstemming met de EN61010 1 2001 elektrische veiligheidsstandaard ontworpen N
23. n 10 35 x 300 mm bottles In addition combinations of both size bottles may be used at the same time e To install the bottle holder carousel Fig 1 slide the bottle wheels 2 onto the rod and tighten the thumb screws Insert the rod into the holder on the right wall Slide the rod into the holder on the left wall of the chamber e To adjust for various bottle lengths Fig 2 loosen the thumb screws on the bottle wheels and reposi tion them along the rod to accommodate the desired bottle length e If skewed bottle positioning is required loosen the thumb screw on only one bottle wheel Fig 2 Slightly rotate the wheel until the reguired angle is obtained Retighten the thumb screw e To insert bottles into bottle clips gently push bottle into the clips Fig 3 coupling sleeve Fig 4 The optional rocker tray Installing the optional rocker tray 0 Remove the bottle holder carousel from the chamber Position the rocker tray in the chamber so that the coupling arm is located on the right side Place the coupling sleeve over the motor drive shaft so that the pins on the inside of the coupling sleeve engage the slots in the motor drive shaft of the chamber O Rotate the sleeve counter clockwise 30 until locked into place o To remove the tray reverse the process Operating instructions Using the Hybridization Oven 0 Place the unit on a level work surface and provide adequate
24. o nado por el eguipo 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 S a puede ser utilizado para operar para mantener y para atender a este producto Advirtiendo Porgue este instrumento puede desarrollar voltaje y corriente suficientes para producir un golpe mortal el cuidado debe ser ejer citado en su operaci n Este instrumento es dise ado de acuerdo con el EN61010 1 2001 est ndar el ctrico de seguridad No obstante debe ser utilizado s lo por operarios adecuadamente capacitados Lea este manual entero antes de utilizar el instrumento y el uso s lo segun las instrucciones El instrumento siempre debe ser utilizado con el plomo de la tierra del cable de alimentaci n moli correctamente a la tierra en la salida de red Utiliza alambre y eguipo el ctricos s lo ilesos especificos para los voltajes que usted utilizar Todo equipo conectado al voltaje alto debe ser de acuerdo con EN61010 1 2001 Mantiene el instrumento tan seco y limpio como posible Enjugue regularmente con un suave el trapo humedo Permita que el instrumento seque completamente antes de uso No opera el instrumento en la humedad extrema encima de 80 Evite condensaci n permitiendo la unidad eguilibra a la temperatura ambiente al O pvi tomar el instrumento de un m s frio a un ambiente m s tibio e Permitir refrigeraci n sufi
25. pera ture In this mode at most a single decimal point is illuminated and the display intensity is steady Changing the setpoint The current setpoint value can be altered using the UP and DOWN buttons while the setpoint is being displayed To change the setpoint from normal mode proceed as follows 0 Momentarily press then release either the UP or the DOWN button The LED intensity will flash to indicate that the displayed value is the current setpoint O Increase or decrease the setpoint value by pressing the UP or DOWN button respectively If either button is held down for more than several seconds the setpoint value will increase or decrease continuously When the desired setpoint is reached wait approxi mately five seconds without pressing either button the display will revert to normal mode showing the actual temperature The new setpoint becomes effective and is stored in the non volatile memory when the display reverts to the normal mode IMPORTANT Reguest a copy of the Hoefer Inc Health and Safety Declaration form before returning the item No items can be accepted for servicing or return unless this form is properly completed If radioactive or biohazardous material has been present within the unit radioactive decontamination and biohazardous clean up as per current Federal State and Local Guidelines and Biological Protocols must be performed BEFORE returning the unit Calibrating
26. re container 2 Rinse briefly in 50 C 0 1X SSC 0 1 SDS 3 Remove this solution and discard in radioactive waste 4 Wash blot three more times in the same solution 5 After the final wash dry the blot on filter paper for 10 minutes This is a good time to quickly pass your hand held radioisotope reader beta or gamma counter over your blot to get a general idea as to the exposure time you will need for the X ray film Hot blots can be placed against film for 20 minutes to 2 hours Otherwise exposure can continue overnight 8 hours 6 Tape the blot to a cardboard backing 7 Cover with plastic wrap to prevent the blots from sticking to the X ray film 8 Place the cardboard containing the blots into the X ray film folder 9 In the darkroom place a sheet of X ray film over the blot s 10 Place an intensifying screen on top of the film 11 Close the film holder and clamp it 12 Store at 70 C The low temperature reduces light scattering and increases the length of exposure time Expose the blot for 20 minutes to 24 hours o pl9 o p20 Protocol Chemiluminescence detection HRP tagged alkaline phosphatase AP probes or antibody conjugates Equipment e Clear plastic cling wrap or clear transparent sheet protector e Hoefer ULTima Pro imaging system with cooled CCD camera e Pipette Reagents Chemiluminescence e ECL or other detection reagent 1 e ECL or other if required
27. room in front of the door for easy access as well as the back for air ventilation Plug the female end of the power cord into the back of the oven To turn on the main power supply press the Power ON OFF switch located on the right side of the control panel to the ON position To adjust the temperature setting press either the UP or DOWN arrow buttons located below the Tempera ture Control display to raise or lower the displayed value degrees Celsius This setting is adjustable from 10 C over ambient up to 99 9 C See Setting the temperature controls page 7 for more details o To turn on the motor for the bottle holder carousel press the Motor Control ON OFF switch to the ON position O To adjust the bottle holder carousel to the desired RPM turn the Motor Control knob clockwise to Increase the RPM and counterclockwise to decrease the RPM Minimum RPM OFF 10 Maximum RPM 18 Mor Comiral ET N Le Tem perrabare Central N pren RPM Control Motor ON OFF Temp setting Main ON OFF Setting the temperature controls Whenever power is applied to the controller the software revision number is displayed for several seconds While the software revision number is being displayed the intensity of the display digits alternates between full and half brightness and on some models the two outside decimals points blink on and off After several seconds the displayed value reverts to the set tem
28. rra alla presa di corrente principale Usa il filo metallico e l apparecchiatura solo intatti elettrici specifici per i voltaggi che lei user Tutta l apparecchiatura collegata all alto voltaggio dovrebbe essere conformemente a EN61010 1 2001 Tiene lo strumento come secco e pulito come possibile Pulire regolarmente con un morbido per spegnere il panno Lasciare lo strumento asciuga completamente prima dell uso Non opera lo strumento nell umidit estrema al di sopra di 80 Evitare la condensazione lasciando l unit eguilibra alla temperatura ambiente quando portare lo strumento da un pi freddo a un ambi ente pi caldo Di permettere raffreddare sufficiente assicura che gli sbocchi dello strumento non sono coperti Viktig Informasjon Norwegian Hvis dette utstyret blir brukt i en m te ikke spesi fisert ved Hoefer Inc beskyttelsen som ha blitt git av utstyret kan bli svekket Dette instrumentet er utformet for innend rs labo ratoriumbruk bare Bare tilbeh r og deler godkjente eller forsynte ved Hoefer Inc kan bli brukt for drive vedlikeholde og betjene dette produktet Varsler Fordi dette instrumentet kan utvikle tilstrekkelig spenning og str m til produsere et d delig sjokk m bli vd bekymring i dets drift Dette instrumentet er utformet i samsvar med EN61010 1 2001 elektrisk sikkerhetsstandard Likevel burde bli brukt det bare av skikkelig utdannede operat rer Les denne hele h ndbo
29. s a risk that bottles will leak and or break due to the internal pressure build up If the bottles are accidentally taken above 70 C with out relieving the pressure DO NOT open the hybrid izer door Turn off the hybridizer and allow it to cool before opening the door and checking the contents of the oven Should the bottles be used above 70 C relieve the pressure by unscrewing and re tightening the cap If using radioactive material see Decontamination page 8 Specifications Power reguirement Net weight Temperature Heating element Temperature display Rotation speed Bottle capacity Dimensions w x h x d Exterior Interior 115 VAC 60 Hz or 230 VAC 50 19 5 kg 43 Ib Ambient 10 C to 99 9 C 1250 watts LCD Off 10 to 18 RPM Twenty 20 35 x 150 mm Ten 10 35 x 300 mm 44 5 x 40 6 x 40 6 cm 17 5 x 16 x 16 in 99 0 27 8 K 27350 14 x 10 75 x 10 75 in Note Always use an even number of bottles and load bottles at opposite points to balance the wheels of the bottle holder carousel This prevents excessive wear of the bearings and drive motor wheels rod Fig 1 Installing bottle holder carousel thumb screw Fig 2 Adjusting for various bottle lengths Fig 3 Inserting bottles Set up instructions Two different bottle sizes are available for use in the hybridization oven The bottle holder carousel can hold up to twenty 20 35 x 150 mm bottles or te
30. spr vn zemn ny k zemi na s ti vyustce Vyu it pouze nepo kozen elektrick dr ty a vybaven pro nap t budete pou vat V echna za zen spojen s vysok m nap t m by m la b t v souladu s EN61010 1 2001 Si ponech n stroje jako such a ist jako mo n Ot ete pravideln s a m kk vlhk m had kem Nech je n stroj nenastaven pln p ed pou it m Nejsou provozov na na n stroj v extr mn vlhkost nad 80 P ede lo kondenzaci o pron jmu jednotky na okoln teplotu nech p i p ij m n n stroj z chladn j do tepl ho prost ed Pro umo n n dostate n chlazen zajistit aby otvory n stroje jsou nevztahuje Vigtig Information Danish Hvis dette udstyr bruges i en m de ikke specifice ret ved Hoefer Inc den beskyttelse som er blevet forsynet af udstyret kan m ske svaekkes Dette instrument er designet for indendors labora toriumbrug bare Bare tilbeh r og del godkendede eller forsynede ved Hoefer Inc kan m ske bruges for drive funk tionsfejl og betjening dette produkt Advare Fordi dette instrument kan udvikle tilstraekkelig spaending og strom at fremstille et dodbringende chok skal pleje bruges i dets drift Dette instrument er designet i overensstemmelse med EN61010 1 2001 elektrisk sikkerhedstandard Alligevel skulle det bruges bare af passende tr nede operat rer L s denne hel h ndbog for bruging instrumentet og brug bare i henhold ti
31. t us directly at 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 supportOhoeferinc com Hybridization technigues There are two main steps to a hybridization reaction 1 hybridizing two strands ot DNA carrying complementary seguences and 2 detec tion of the hybridized DNA Proximity of the DNA strands determines the freguency of the binding event and therefore success is depen dent on the DNA concentrations Since the concentration of the target nucleic acid is usually unknown it is an excess of labeled probe DNA that drives forward the hybridization reaction This is simply increasing the chances of a probe encountering a target But with an enormous amount of probe present in solution or on the surface of a membrane the back ground signal will also be enormous The typical approach to correct for excess background on a membrane or slide hybridization is to wash in a low salt buffer as this favors the dissociation of unbound probe from the membrane slide and from non complementary DNA In solutions a probe can be enzymatically degraded by using a single strand specific nuclease o pll Fig 5 o p12 Mechanisms of nucleic hybridization Hybridization occurs with a process called nucleation whereby the two separate nucleic acid strands come into close proximity of each other A duplex region is formed where a minimum of thr
32. the temperature reading The hybridizer is calibrated at the factory Hoefer Inc recommends temperature recalibra tion be performed at the factory as recalibration by the user may void the warranty Obtain a RA Return Authorization number from Hoefer Inc prior to returning a unit to the factory Decontamination Bottles and caps e Soak items in a diluted detergent solution overnight e Remove items from detergent and rinse with distilled water e If items are still contaminated gently scrub with an abrasive cloth or brush If necessary continue to soak items in the detergent solution for a longer period of time Oven chamber e The oven chamber and drip pans may be decon taminated by wiping clean with a decontaminating agent then repeat with distilled water These decontamination methods may not remove all contaminants Refer to Federal State and Local Guidelines and Biological Protocols to assure decontamination Bottle care Hybridization bottles are made of lead free borosilicate glass which provides excellent long term reliability Each bottle includes a polypro pylene cap and Teflon seal e It is important to check your bottles regularly for chips stress fractures and cracks If these occur the bottle must be discarded e Between use store bottles either in a suitable rack or with caps attached This will protect the bottle and seal e Wear protective gloves to protect your hands in t
33. under the carousel allows easy clean up of spilled media A touch sensitive keypad and microprocessor provide accurate temperature control from 10 C over ambient up to 99 9 C The keypad is located just below a large LED readout Once the desired hybridization temperature is entered the readout displays the current temperature inside the chamber The chamber environment is calibrated at 68 0 C at this temperature the accuracy is 0 5 C An internal circulating fan assists unitorm distribution of heating through out the chamber Two different hybridization bottle sizes are available for use within the hybridizer using the standard bottle holder carousel provided with the unit The bottle holder carousel can hold up to twenty 20 35 x 150 mm bottles or ten 10 35 x 300 mm bottles In addition combinations of both size bottles may be used at the same time WARNING There may be build up of pressure within the hybrid ization bottles when they are taken from ambient to hybridization temperature To help in relieving some of this pressure preheat the solutions and bottles Also be sure bottles are opened at the same temperature at which the hybridization was done Do not allow the bottles to cool before opening To assure hybridization bottles remain leakproof and pressure proof do not hybridize at temperatures above 70 C If temperatures above this are used without first relieving the pressure within the bottle there i
34. voi kehitt riit t v j nnitteen ja virran tuottaa kuolettavan j rky tyksen huolta t ytyy harjoittaa toiminnossaan T m v line suunnitellaan EN61010 1 2001 s hk turvallisuusstandardin mukaisesti Silti pit isi k yt t vain ohi oikeasti koulutetut k ytt j t Lue t m kokonainen manuaalinen ennen v linett ja k ytt vain ohjeiden mukaan V linett t ytyy k ytt aina valtanuoran maaly ijyst perusti oikein maadoittaa s hk verkkoau kossa K ytt vain undamaged s hk metallilankaa ja varusteita t sm llinen j nnitteille k ytt Kaikki varusteet yhdistetty korkeaan j nnitteeseen pit isi olla EN61010 1 2001IN mukaisesti Pit v lineen yht kuiva ja puhdas kuin mahdol linen Pyyhi s nn llisesti pehme ll kostealla kankaalla Anna v line kuivua t ysin ennen k ytt Ei k yt v linett extreme ilmankosteudessa 80 n yl puolella V lt tiivistymist antamalla yksik n eguilibrate ymp r iv n l mp tilaan kun ottaminen v line kylmempi l mpim mp n ymp rist n Sallia riitt v n j hdytt minen varmistaa ett v lineen ilmarei t peitet n O piii Information Importante French e 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 e Cet instrument est con u pour l usage de labora toire int rieur seulement e Seulement les accessoir
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