Hoefer® HE 100 SuperSub - GE Healthcare Life Sciences
Contents
1. 0 Pop the lid out of the connectors by resting the thumbs on both posts protruding through the lid by each electrode connector while lifting up under the lid with both index fingers Running platform Buffer chamber inlet side Internal buffer circulation feeds from this end Coolant circulation ports 2 for extemal cooling circuit 7 Electrode post 2 Caution Ethidium bromide is a known mutagen Always wear gloves when handling Table 1 Volume of agarose required for different gel sizes Operating Instructions Agarose gels are first cast in the gel casting tray The running tray is then trans ferred to the platform of the unit samples are loaded into wells created by a comb and the sample is electrophoretically separated The fluorescent dye ethid ium bromide can be added to the ga or electrophoresis buffer or both in order to track separation progress After electrophoresis the gel may be stained and pho tographed blotted or dried for autoradiography 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 levelling feet Casting the gel Prepare the solutions 1 Prepare about 1 5 liters of running buffer Approximately 300 ml of buffer is required for the gel and 1 2 liters for the buffer chamber Refer to p 13 for recipes of2. 5 1 526 15 2 80 6047 13 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 ref erence well 14 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 15 DNA detection DNA can be detected either by the fluorescence of bound ethidium bromide or by autoradiography of radio labeled DNA Ethidium bromide 0 5 ug ml is often added to running buffer to monitor sam ple progress because the dye s fluorescence reveals DNA under a UV lamp To check band location turn off the power supply and remove 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 solu tion 0 5 ug ml H 0 for 15 to 60 minutes and then view or photograph the sample on a UV transilluminator Note Minimize the staining time to prevent small nucleic acid fragments from diffusing out of the gel 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
3. SuperSub and then fill with deionized water Place the unit on a magnetic stirrer and allow it to circulate to rinse all internal surfaces See Prepare the unit step 2 Stop circulation and empty the unit To empty the circulation chamber turn the unit upside down outlet chamber facing down Allow the liquid to drain Note If a thorough cleaning is required disassemble the base by removing the two fittings and six screws on the base and sides 11 Troubleshooting Sample well deformed Y Y Y 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 along a straight path Y Y Y If comb is warped replace If running tray is warped replace Cool agarose to 50 C to prevent the tray from warping Circulate the buffer at about 100 ml min This is the slowest speed on most magnetic stirrers Double banded pattern Y Y Y Make sure the comb is vertical during casting 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 through the gel Avoid a temperature gradient in the gel the cooling temp
4. buffer chamber in only one orientation The fully shielded 4 mm safety plugs connect to acompatible DC power supply Running trays All running trays are 20 cm wide Three tray lengths are avail able 15 20 and 25 cm The two shorter trays each can hold one or two combs and the 25 cm long tray can hold up to four combs The tray has an anchor notch at each end to help keep the gel in place during transport or when high er circulation rates are required Gels can be cast either by setting the running tray into a casting tray or by taping the ends of the tray All trays are UV trans parent for convenient visualizing of the stained results Casting tray Using the appropriate length casting tray eliminates the taping step Agarose should be cooled to 50 C before pouring to prevent warping of the tray Combs Available combs range in thickness from 1 to 6 mm and have 6 12 15 20 30 or 36 wells Preparative combs with 2 or 3 reference wells are also avail able The 20 well combs are designed for loading wells with a multi channel pipetter Well Locating Decal Place on the running platform under the running tray to see wells more easily while loading samples Figure 1 Horizontal submarine unit main components Gel casting kits combs and comb backs may be ordered separately the ordering sec tion tabulates all comb sizes and accessories Color coded leads connect electrodes in the unit base to the power supply
5. consecuencias relacionadas cualquiera que sea la causa que se deban a la utilizaci n defectuosa e incorrecta del producto Copyright 1997 Amersham Biosciences AB Reservados todos los derechos No est permitida la reproducci n ni el almacenaje en un sistema de recuperaci n ni la transmisi n de parte alguna de esta publicaci n sin la autorizaci n por escrito de la empresa Wichtige Benutzerinformationen F r ein vollstandiges Verstandnis und eine Deutsch sichere Handhabung dieses Produktes ist es notwendig da der Benutzer dieses Handbuch vollst ndig durchliest A A Wenn Sie Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Ein Ausrufezeichen in einem gleichseitigen Dreieck soll den Benutzer auf die Anwesenheit wichtiger Betriebs und Wartungsanweisungen in der dem Gerat beiliegenden Dokumentation hinweisen Ein Blitzsymbol in einem gleichseitigen Dreieck soll den Benutzer auf die Gefahr anliegender Hochspannungen hin weisen Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences beh lt sich das Recht vor die Spezifikationen ohne vorhergehende Ank ndigung zu ndern Gew hrleistung and Haftung Amersham Biosciences garantiert da das gelieferte Produkt sorgf ltig auf die Einhaltung der ver ffentlichten Spezifikationen getestet wurde Die in den Lieferbedingungen n her erl uterten Gewahrleistungsanspruche gel ten
6. light and 40 transparent to 254 nm light If you place the gel on the transilluminator cut off the ridges formed by the grooves in the running tray so that the gel lies flat Do not damage the transil luminator surface trim both ends of the gel with a spatula while still in the tray lift away the ridges and then slide the gel onto the transilluminator For view ing 302 nm light is recommended for both acceptable sensitivity and reduced photoknicking To reduce the background fluorescence of unbound ethidium bromide the gel can be destained by soaking it for 5 minutes in 0 01 M MgCl or for 1 hour in 0 001 M MgSO Destaining makes it easier to detect small quantities less than 10 ng of DNA Sambrook p 6 15 Transfer Before transfer trim off both ridges at both ends of the gel to ensure even gel contact with the membrane 16 Bibliography and References General Reviews of DNA RNA Electrophoresis Ausubel et al eds Current Protocols in Molecular Biology Greene Publishing and Wiley Interscience New York 1993 Rickwood D and Hames B D Gel Electrophoresis of Nucleic Acids 2nd ed IRL Press Ltd 1990 Sambrook J Fritsch E F and Maniatis T Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press 1989 RNA Separation Methods Lavialle C Reuveni Y Thoren M and Saltzman N P Molecular interaction between Simian virus 40 DNA and Escherichia coli RNA polymerase Exam
7. sample loading buffer Ys of the final volume is loading buffer see p 12 Mix and load each sample into a well with a micro pipet taking care to avoid puncturing the well bottom or entrapping bub bles in a well Place the lid on the unit so that the cathode black lead is at the end nearest the sample wells Nucleic acid samples migrate toward the anode Connect the color coded leads red to red and black to black to an approved power supply such as the EPS 600 or the Hoefer EPS 24200 and set the voltage and timer if available Starting point guidelines for DNA separations Hind III digest of lambda phage 2 27 kb 0 5X TBE buffer are 200 V constant voltage a maximum current set ting of 200 mA and the circulator set to 20 C This separation typically requires 3 4 hours Important Electrophoresis should be under way several minutes before start ing buffer circulation to prevent samples from being washed out of the wells Once circulation is established set the stirrer to the lowest speed about 100 ml min If the stirrer is too powerful try placing two glass plates on the stirrer to decrease the strength of the magnetic field 10 After the separation 1 2 Turn off the power supply disconnect the leads and remove the lid 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 If the gel is still in the runnin
8. three commonly used electrophoretic running buffers Optional Prechill the buffer either before pouring into the SuperSub or after by connecting the heat exchanger to an extemal circulator bath 2 Prepare the sample loading buffer Refer to page 14 for a recipe and tabulat ed volume capacity for each comb size 3 Prepare agarose solution s Dissolve agarose in running buffer 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 facili tate observation of separation progress during electrophoresis Approximate volume of agarose ml for various gel thicknesses mm Tray size cm 3 4 5 6 20x15 90 120 150 180 20 x 20 120 160 200 240 20 x 25 200 250 300 Casting the gel Prepare the combs 1 Align the three slots in the comb with the loosened thumb screws on the comb back Tighten the screws until the comb is just supported 2 Place the comb assembly into a set of slots on the Comb back l Casting tray and adjust the comb so that the bot i tom is 1 0 mm from the running tray Tighten the screws to secure the comb To run twice as po many samples on the 15 and 20 cm trays prepare two combs Prepare up to four combs for the 25 Screws cm tray Pour the gel 1 Press the running tray into the casting tray The running tray should lay flush against the bottom of
9. 17 1324 01 17 1322 01 80 6297 36 80 6312 12 80 6012 36 19 0200 00 19 0600 00 80 6274 18 80 6274 37 18 1102 77 18 1102 78 80 6246 82 80 6247 01 80 6247 20 80 6245 11 80 6245 30 80 6224 78 80 6224 97 80 6077 34 80 6297 36 22845 Rev E 5 97 Hoefer HE 100 SuperSub Submarine Electrophoresis Unit User Manual uF W N SuperSub Function and Description Specifications Important Information Unpacking and Inventory Operating Instructions Care and Maintenance Troubleshooting Notes Buffers and Volumes Bibliography and References Customer Service Information Important user information a Please read this entire manual to fully under Eng lish stand the safe and effective use of this product The exclamation mark within an equilateral triangleis intended to alert the user to the presence of important oper ating and maintenance instructions in the literature accom panying the instrument The lightning symbol within an equilateral triangle is intended to alert the user to the risk of exposure to high voltages Should you have any comments on this manual we will be pleased to receive them at Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences reserves the right to make changes in the specif
10. 45 80 36 1 0 3 0 80 6045 99 36 1 5 3 0 80 6046 18 36 3 0 3 0 80 6046 37 1 2 1 0 176 5 80 6046 75 1 2 1 5 176 5 80 6046 94 1 2 3 0 176 5 80 6047 13 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 Printed in the USA 18 Casting and running trays HE 100 Gel Casting Kit 20x15 cm 20x20 cm 20x25 cm Includes casting and running trays HE 100 Casting Tray 20x15 cm 2020 cm 2025 cm HE 100 UVT Running Tray 20x15 cm 2020 cm 20x25 cm Reagents Ethidium bromide Agarose NA Agarose prep Ficoll 400 Bromophenol Blue Tris EDTA disodium salt Boric acid Manuals and Technical Bulletin User manual Service manual Amersham Biosciences Technical Bulletin 137 Companion products EPS 200 power supply EPS 600 power supply EPS 2A200 power supply 115 V 230 V MultiTemp Ill Thermostatic Circulator 115 V 230 V ImageM aster VDS DU 115 V 60 Hz DE 230 V 50 Hz DJ 100 V 50 60 Hz MacroVue UV 20 Translluminator 115 V 230 V MacroVue UV 25 Transilluminator 115 V 230 V Photoman Polaroid Direct 10 ml 100 g 50 g 100 g 10 g 500 g 100 g 500 g 80 6048 84 80 6049 03 80 6049 22 80 6048 08 80 6048 27 80 6048 46 80 6047 51 80 6047 70 80 6047 89 17 1328 01 17 0554 02 80 1130 07 17 0400 01 17 1329 01 17 1321 01
11. SuperSub Function and Description The Hoefer HE 100 SuperSub Submarine Electrophoresis Unit electrophoreti cally separates nucleic acid fragments in agarose gel The technique is simple and sensitive and by varying the agarose concentration fragments over a large size range can be separated Fragments stained with ethidium bromide can be easily visualized under UV light All gels are 20 cm wide Gel casters for three lengths 15 20 and 25 cm are available Gel thickness can range from 3 to 7 mm The gel is first cast in the running tray with or without a casting tray After the gel sets the running tray is transferred to the horizontal unit Two features contribute to the success of higher voltage runs without loss of res olution 1 The built in buffer circulation system activated by a magnetic stir rer maintains uniform buffer pH and ionic strength as well as uniform tempera ture at the gel surface which can be critical in certain applications such as gly oxylated RNA separations 2 A heat exchanger under the gel platform controls buffer temperature when attached to an external cooling system Temperature control is especially important when using field inversion techniques to sepa rate large DNA fragments Amersham e Biosciences Specifications Max voltage Max wattage Max amperage Max operating temperature Max buffer volume Gel size Environmental operating conditions Installation category Pollutio
12. are the unit 1 Optional cooling Connect the heat exchanger to a thermostatic circulator such as the MultiTemp III Slide hose clamps 4 total onto each end of two lengths of 8 or 9 mmi d approx 38 vinyl or silicone tubing Attach one end of each length of tubing to a heat exchanger port Attach the free ends of each length of tubing to the circulator ports one to the inlet and the other to the outlet Secure the connections with the hose clamps Important D Use only water or 50 50 water ethylene glycol as a coolant Never use a com mercial antifreeze or any alcohol based mixture or irreparable damage to the heat exchanger will result D Do not connect the heat exchanger to a water tap or any other source where the water pressure is unregulated A starting point temperature setting for the circulator is 20 C for electrophore sis at or below 200 V Adjust as necessary for variables such as ambient temper ature changes in power output and circulator efficiency If accurate tempera ture control is critical measure the temperature and adjust as necessary Optional After establishing buffer circulation step 2 below run the circulator to prechill the buffer for about 30 minutes before starting electrophoresis Establish buffer circulation Locate the inlet buffer chamber at the right side of the unit if the coolant ports are in the back The inlet is a groove along the chamber floor Slowly add buffer to this cha
13. erature should be set no lower than 20 C at 200 V Poor band resolution Y je Sos Ss SON Add Ficoll glycerol or sucrose to the sample loading buffer to ensure that the sample layers on the bottom of the well Ficoll is the recommended agent Make sure the sample is completely dissolved Reduce the voltage Reduce the sample concentration Reduce the sample volume Be sure there is at least 1 mm of gel below the bottom of the comb to pre vent samples from leaking out the bottom of the well Reduce salt concentration of the sample Check enzyme activity may require longer digestion or a different restric tion buffer Prepare fresh sample if you suspect nuclease contamination Choose agarose with a low endosmosis value Table 2 Agarose concentra tions for separating DNA fragments of various sizes Note RNA samples usually require longer runs or buffers that are easily depleted so it is neces sary to circulate the buffer 12 Notes Buffers and Volumes Agarose gel electrophoresis notes Agarose gel electrophoresis can be used to separate DNA fragments as small as 0 1 kb or less Polyacrylamide gels are usually used for fragments smaller than 1 kb DNA mobility Suggested agarose concentration for separating fragments of various sizes is given in Table 1 below Other factors affecting separation results include the running buffer the voltage setting the temperature and the presence of eth
14. essure is unregulated Cool the agarose to 50 C before pouring into the casting kit to prevent plastic parts from warping Do not operate with buffer temperature above 45 C All plastic parts are rated for 45 C continuous duty Circulate coolant through the heat exchanger during elec trophoresis to minimize heating Overheating will cause irreparable damage to the unit Faire circuler seulement de l eau ou 50 50 d eau et d thyl ne glycol dans l changeur vertical cirulation d eau Ne jamais utiliser d anti gel ou tout autre solvant organique avec cet instrument Les solvants organiques causeraient des dom mages irr parables l appareil Ne pas connecter l changeur vertical circula tion d eau un robinet ou quelque source de refroidissement dont la pression n est pas r guli re Refroidir l agarose entre 50 C avant de la verser dans l unit de moulage afin d viter que les pi ces en plastique ne se d forment Ne pas utiliser avec un tampon une temp ra ture au dessus de 45 C Toutes les pi ces en plas tique sont pr vues pour r sister une temp rature constante de 45 C Faire circuler l eau dans l changeur vertical durant l lectrophor se pour minimiser l chauffement afin d viter des dommages irr parables l instrument If this equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be
15. g tray staining will take longer Visualize the gel on a transilluminator Transfer the gel using the UV transparent running tray For maximum visualization place the gel directly on the transillumi nator see step 4 To reduce the background fluorescence of unbound ethidium bromide the gel can be destained by soaking it for 10 min in distilled water or TBE buffer Destaining makes it easier to detect quantities of DNA less than 50 ng Remove the gel from the running tray by gently pulling up on the outer edges of the gel If the gel is fragile run a spatula or the Hoefer Wonder Wedge between the gel and tray to free the gel anchor Clean the unit as described below Care and Maintenance 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 30 or concentrated acids over 25 Adhesive from the sealing tape may be removed from the running tray by gently wiping the surface with kerosene To remove DNase and RNase contamination fill the unit with 3 hydrogen peroxide H202 soak for 10 minutes and circulate the solution see Prepare the unit step 2 Rinse thoroughly with DEPC treated autoclaved deionized water Sambrook et al 1 7 40 The unit is resistant to all common electrophoresis buffers but we recommend a thorough washing after each use 1 2 Rinse the
16. i riserva il diritto di apportare modifiche ai dati tecni ci senza preavviso Garanzia e responsabilit Amersham Biosciences garantisce che prima della consegna il prodotto stato collaudato a fondo per soddisfare i requisiti specificati La garanzia inclusa nelle condizioni di consegna risulta valida solamente se il prodot to stato installato ed utilizzato nel rispetto delle istruzioni fornite da Amersham Biosciences Amersham Biosciences non potr essere ritenuta responsabile di incidenti o danni consequenziali inclusi ma non limitati a perdite di profitti manca to guadagno perdite di affari difetti di funzionamento e relative espo sizioni dovuti ad un utilizzo non corretto del prodotto Copyright 1997 Amersham Biosciences AB Tutti i diritti riservati Nessuna parte della presente pubblicazione pud essere riprodotta conservata in sistemi di gestione dati o trasmessa in alcun forma n per nessuno scopo senza autorizzazione scritta del produttore
17. ica tions without prior notice Warranty and Liability Amersham Biosciences guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the product has been installed and used according to the instructions supplied by Amersham Biosciences Amersham Biosciences shall in no event be liable for incidental or conse quential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Copyright 1997 Amersham Biosciences AB All rights reserved No part of this publication may be reproduced stored in aretrieval system or transmitted in any form by any means without permission in written form from the company Renseignements importants d utilization Pour une bonne compr hension et une utilisa tion en s curit maximale il convient delire enti rement ce manuel A A Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser Fran ais Dans la documentation qui accompagne l instrument un point d exclamation dans un triangle quilat ral a pour but d attirer l attention de l utilisateur sur des instructions impor tantes de fonctionnement ou de maintenance Le symbole de l clair dans un triangle quilat ral a pour
18. idi um bromide Special agaroses are available that can extend resolution ranges Agarose Effective range of resolution of linear DNA fragments kb 0 5 30 gt 1 0 0 7 12 gt 0 8 1 0 10 gt 0 5 1 2 7 gt 0 4 1 5 3 gt 0 2 Current Protocols in Molecular Biology p 2 5 2 1993 A common standard is a Hind III digest of lambda phage which gives eight frag ments ranging in size from 0 1 to 23 kb pairs The bands are well resolved when run 3 hours on a 20 cm long 1 agarose gel in 0 5X TBE gel at 200 V RNA mobility RNA can also be separated on the basis of size To avoid irregularities due to sec ondary structure RNA is denatured either before or during electrophoresis For example RNA fragments previously denatured with glyoxal and dimethylsulfox ide can be separated on neutral agarose gels or RNA can be fractionated on agarose gels containing methylmercuric hydroxide or formaldeh yde Request Hoefer Technical Bulletin 137 Low Formaldehyde Denaturing RNA Gel Electrophoresis Protocol for Northern Blotting with the TE 80 TransVac Vacuum Transfer Unit for an example of RNA electrophoresis Running buffers for DNA in agarose gels Recipes for the two most commonly used running buffers for DNA electrophore sis are listed below The ionic strength of these buffers is appropriate for the application do not adjust the pH of these buffers once they are prepared accord ing to the recipe The buffering capacity
19. impaired Only accessories and parts approved or sup plied by Amersham Biosciences may be used for operating maintaining and servicing this product Si l instrument n est pas utilis en conformit avec les recommandations du fabriquant les protec tions de s curit qui quipent cet appareil peuvent tre rendues in fficaces Seulement les accessoires et pi ces detach es approuv s ou fournis par Amersham Biosciences sont recommand s pour l utilisation l entretien et r pa ration de cet appareil Unpacking and Inventory Unwrap all packages carefully and compare contents with the packing list mak ing sure all items arrived If any part is missing contact your local Amersham Biosciences 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 Buffer chamber Both platinum electrodes are located at the outer lower edges of each buffer chamber so that bubbles generated during electrophoresis do not build up near the gel Below the running platform are housed both an internal buffer circulation system that can be driven by a magnetic stirrer and a heat exchanger that can be attached to an external circulator bath Safety lid The safety lid houses the electrode connectors and leads It fits on the
20. mber until the spiral conduit is filled allowing air bubbles to escape If necessary tip the unit so that the inlet chamber is lower than the outlet chamber Note A large number of bubbles in the pumping chamber will interfere with circulation efficiency but a few remaining should not Place a magnetic stirrer on a leveling plate or other level surface then place the SuperSub on the magnetic stirrer Place the spirit level on the running platform and level the unit Turn on the magnetic stirrer and move the unit as necessary until the stir bar spins freely Adjust the stirring speed to achieve the required circulation rate the maximum rate for prechilling and the lowest rate during electrophoresis Circulation at the maximum rate is not recommended for electrophoresis because high flow rates may result in uneven cooling and turbulence that could wash sample out of the wells Caution Wear UV safety goggles and protect skin when using a UV lamp Important If running two sets of samples in the same gel monitor the run closely and stop elec trophoresis when the marker dye approaches the wells in the center Separating the sample Refer to the Notes Buffers and Volume section for additional informa tion and guidelines 1 Turn off the magnetic stirrer to stop buffer circulation Optional To more easily see wells for sample loading place the Well Locating Decal on the running platform printed side down to preven
21. n degree Dimensions w x x d includes electrode posts Product certifications 500 V 40 W 500 mA 45 C 950 1175 ml depending on gel size 20 cm wide X15 20 or 25 cm long 3 7 cm thick Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Il 2 25 x 32 x 8 5 cm 9 9x12 63 4 in EN61010 1 UL3101 1 CSA C22 2 1010 1 CE This declaration of conformity is only valid for the instrument when it is p used in laboratory locations p Used as delivered from Amersham Biosciences except for alterations described in the User Manual and p connected to other CE labeled instruments or products recommended or approved by Amersham Biosciences Important information The safety lid must be in place before con necting the power leads to a power supply Turn all power supply controls off and dis connect the power leads before removing the safety lid Informations importantes Le couvercle de s curit doit tre en place avant de brancher les prises au g n rateur Eteindre le g n rateur et d brancher les prises avant d enlever le couvercle de s curit Circulate only water or 50 50 water ethyl ene glycol through the heat exchanger Never introduce anti freeze or any organic sol vent into any part of the instrument Organic solvents will cause irreparable damage to the unit Do not connect the heat exchanger to a water tap or any coolant source where the water pr
22. n partie sont strictement interdits sans autorisation pr alable crite de la soci t Informaci n importante para el usuario Para comprender el producto y utilizarlo con seguridad es necesario leer este manual en su totalidad A A Si desearan hacer alg n comentario sobre este manual tengan la amabili dad de remitirlo a Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se reserva el derecho a modificar las especificaciones sin previo aviso Espa ol El signo de admiraci n en un tri ngulo equil tero en el manu al advierte al usuario sobre la presencia de instrucciones importantes de operaci n y mantenimiento del aparato El s mbolo del rayo en un tri ngulo equil tero alerta al usuario sobre el riesgo de exposici n a altas tensiones Garant a y responsabilidad Amersham Biosciences garantiza que el producto entregado ha sido probado a fondo para comprobar el cumplimiento de las especificaciones publi cadas La garant a incluida en las condiciones de entrega s lo es v lida si el producto se ha instalado y utilizado de acuerdo con las instrucciones entregadas por Amersham Biosciences Amersham Biosciences no ser responsable bajo ning n concepto de da os directos o indirectos incluyendo sin limitaci n la p rdida de beneficios la p rdida de ingresos la p rdida de oportunidades de negocio la p rdida de utilizaci n y otras
23. nur dann wenn das Produkt gem den von Amersham Biosciences gelieferten Anweisungen installiert und benutzt wurde Amersham Biosciences Ubernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf GewinneinbuBen Einkommensverluste entgangene Gesch ftsabschl sse Verlust der Gebrauchsfahigkeit oder andere Verluste die wie auch immer durch eine fehlerhafte oder unsachgem e Verwendung des Produkts verursacht wurden Copyright 1997 Amersham Biosciences AB Alle Rechte vorbehalten Die vorliegende Ver ffentlichung darf nur mit vorhergehender schriftlicher Genehmigung durch das Unternehmen vervielf ltigt in einem Abrufsystem gespeichert oder in irgendeiner Form oder mit irgendwelchen Mitteln bertragen werden Informazioni importanti per l operatore Per un utilizzo scuro del prodotto leggere attentamente l intero contenuto del presente manuale A A Si prega di inviare eventuali commenti al presente manuale a Italiano Il punto esclamativo all interno di un triangolo equilatero indica all operatore la presenza di importanti istruzioni di funzionamento e manutenzione nella documentazione allega ta al prodotto Il simbolo del fulmine all interno di un triangolo equilatero indica all utente la presenza di un rischio di esposizione ad alte tensioni Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences s
24. o the recipe Modified from Sambrook J Molecular Cloning A Laboratory Manual p B 23 1989 See also Current Protocols in Molecular Biology p A 2 1 1993 13 Sample loading buffer Loading buffer 5X 25 Ficoll 400 0 25 Bromphenol blue 10 ml Deionized H2O to 7 0 ml Ficoll 400 Amersham Biosciences 2 59 Bromphenol blue FW 691 9 25 0 mg Deionized H2O to 10 0 ml Add to sample in proportion so that 5 of the final volume is loading buffer 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 and dragging effects caused by comigration with smaller nucleic acids Table 3 wells sample vol per Sample volume no thickness mm width mm 1mm depth pl Code no required for various 12 1 0 13 7 13 7 80 6044 47 combs 12 1 5 13 7 20 5 80 6044 66 12 3 0 13 7 41 1 80 6044 85 20 1 0 7 3 7 3 80 6045 04 20 1 5 7 3 10 8 80 6045 23 20 3 0 7 3 21 8 80 6045 42 30 1 0 4 5 4 5 80 6045 61 30 3 0 4 5 13 5 80 6045 80 36 1 0 3 0 3 0 80 6045 99 36 1 5 3 0 4 5 80 6046 18 36 3 0 3 0 8 9 80 6046 37 1 2 1 0 175 6 5 1 175 6 5 1 80 6046 75 1 2 1 5 175 6 5 1 175 6 5 1 80 6046 94 1 2 3 0 175 6
25. objet d attirer l attention de l utilisateur sur un danger d ex position la hautetension Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se r serve le droit d effectuer des modifications de ces sp cifications sans aucun pr avis Garantie et responsabilit Amersham Biosciences garantit l utilisateur que le produit livr a subi avec succ s tous les essais pr vus pour s assurer qu il est conforme aux sp cifi cations et normes en vigueur La garantie incluse dans les conditions de livraison n est valable que si le produit a t install et utilis conform ment aux instructions fournies par Amersham Biosciences La soci t Amersham Biosciences ne sera en aucun cas responsable de tout dommage caus directement ou indirectement par toute utilisation incor recte ou non approuv e du produit ou d coulant de cette utilisation y compris toute perte de b n fice ou de recettes toute perte de perspectives commerciales tout emp chement d utilisation et tout autre risques ayant un rapport avec l utilisation du produit mais sans aucune limitation quant la nature de ces dommages Copyright 1997 Amersham Biosciences AB Tous droits r serv s La reproduction le stockage dans un syst me de r cup ra tion d informations ou la transmission sous quelque forme que ce soit et par quelque moyen que ce soit de la pr sente publication en totalit ou e
26. of both TBE and TPE is usually suffi cient so that buffer circulation is unnecessary Circulation may be required dur ing runs longer than 3 hours or when using TAE buffer Important 1 10X Tris borate EDTA TBE stock buffer 0 90 M Tris 0 90 M boric acid 20 mM EDTA pH 8 2 1000 ml Do not adjust the pH of these k buffers once they are prepared Tris base FW 121 1 0 90 M 109 0 g according to the recipe Boric acid FW 61 8 0 90 M 55 6g EDTA solution 0 5 M pH 8 0 soln 3 0 20 M 40 0 ml Deionized H20 to 1000 0 ml Stir Do not adjust pH BEFORE USE DILUTE EITHER TO 0 5 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 90 mM Tris base 90 mM boric acid and 2 mM EDTA 2 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 y Acetic acid 85 0 20 M 11 4 mi EDTA solution 0 5 M pH 8 0 soin 3 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 3 EDTA solution ethylenediamine tetraacetic acid 0 5 M pH 8 0 100 ml NagEDTA 2H20 FW 372 2 0 5 M 18 6 g Deionized H20 to 70 0 ml NaOH 10 M to pH 8 0 5 0 ml Deionized H2O to 100 0 ml Important Do not adjust the pH of these buffers once they are pre pared according t
27. ple of electrophoresis of RNA treated with glyoxal and dimethyl sulfoxide J Biol Chem 257 1549 1557 1982 Lehrach H Diamond D Wozney J M and Boedtker H RNA molecular weight determina tions by gel electrophoresis under denaturing conditions a critical re examination Biochem 16 4743 4751 1977 Kroczek R A and Siebert E Optimization of Northern Analysis by Vacuum Blotting RNA Transfer Visualization and Ultraviolet Fixation Anal Biochem 184 90 95 1990 Field Inversion Gel Electrophoresis Birren B W Lai E Hood L and Simon M I Pulsed field gel electrophoresis techniques for separating 1 to 50 kilobase DNA fragments Anal Biochem 177 282 286 1989 Bostock C S Parameters of field inversion gel electrophoresis for the analysis of pox virus genomes Nucl Acids Res 16 10 4239 4252 1988 Carle G F Frank F and Olson M V Electrophoretic separations of large DNA molecules by periodic inversion of the electric field Science 232 65 68 1986 Crater G D Gregg M R Holzwarth G Mobility surfaces for field inversion gel elec trophoresis of linear DNA Electrophoresis 10 310 315 1989 Denko N Giaccia A Peters B Stamato T D An asymmetric field inversion gel elec trophoresis method for the separation of large DNA molecules Anal Biochem 178 172 176 1989 Customer Service Information Technical Service and Repair Amersham Biosciences offers complete
28. t the lines from becoming defaced First fold the tabs down at the four corners and then place making sure that the side marked is next to the positive electrode Note Gels cast in the 25 cm tray may not coincide perfectly with the shaded areas on the Decal Alternatively if your wells are always in the same location apply a length of red tape across the running platform to mark that area Transfer the running tray with the gel to the running platform orienting it so that the sample will run to red That is place the sample wells at the cathode end which is indicated by a black dot Add buffer until the gel is submerged under 1 mm of liquid This is the opti mal depth too much buffer will cause excessive heat to be generated and too little buffer will cause excessive turbulence around the gel Optional Although this technique is not recommended migration progress can be monitored by either adding 0 5 ug ml final conc of ethidium bromide to the running buffer now or adding 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 migra tion slightly Detection by this method is not as sensitive as staining and viewing on atransilluminator See the DNA detection section for more details Load the samples Add the sample to 5X
29. technical support for all our products If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Amersham Biosciences representative Important Request a copy of the Amersham Biosciences Health and Safety Declaration Form before returning the item No items can be accepted for ser vicing or return unless this form is properly completed Ordering Information Qty Code No HE 100 SuperSub Submarine Hectrophoresis Unit basic Includes spirit level 1 80 6043 90 Order gel casting kit comb and comb back separately HE 100 SuperSub Submarine Hectrophoresis Unit complete 1 80 6043 71 Includes basic unit 1 0 mm thick 20 well comb and comb back 20x25 cm running plate and 20x25 cm casting tray Accessories and replacement parts Buffer chamber assembly only 1 80 6044 09 Comb back for HE 100 combs with 3 screws 1 80 6046 56 Lid with power cables 1 80 6048 65 High voltage leads set 1 80 6177 09 Mylar sealing tape 1 roll 1 80 6124 65 Quick fit coupler body female to fit 3 8 i d tubing 2 80 6115 15 Quick fit coupler body male to fit 3 8 i d tubing 2 80 6115 53 Tubing for coolant silicone 8 mm i d 12 mm o d 4m 80 1106 56 HE 100 Combs wells no thickness mm width mm 12 1 0 13 7 80 6044 47 12 1 5 13 7 80 6044 66 12 3 0 13 7 80 6044 85 20 1 0 7 3 80 6045 04 20 1 5 7 3 80 6045 23 20 3 0 7 3 80 6045 42 30 1 0 4 5 80 6045 61 30 3 0 4 5 80 60
30. the casting tray Alternative casting assembly Tape both ends of the running tray making sure the tape is high enough to contain the agarose 2 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 will allow 1 mm of space between the comb bottom and the running tray Remove the level and the comb assembly 3 Pour the agarose solution cooled to 50 C into the casting assembly Orient the comb assembly so that the comb side faces the gel Fit the comb assembly into the slots The 20 cm and 15 cm trays can hold two combs one at the cathode end indicated by the black dot and one in the center The 25 cm tray can hold 4 combs 5 Allow a minimum of one hour for the gel to set then remove the comb careful ly partially lift and slightly tilt the comb at one end and then 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 Lift the running tray out of the casting tray or remove the tape from the ends of the running tray if no casting tray was used 6 Remove any agarose adhering to the underside of the running tray Optional Store the cast gel in running buffer for up to two days at 4 C Note If the cooling option is used frequently it is convenient to attach Quick fit fittings to the tubing The valves in these fittings prevent coolant spillage Prep
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