HE 33 Mini Submarine Electrophoresis Unit User Manual
Contents
1. be used for operating maintaining and servicing this prod uct Ame A rsham B b b b b b b Avant la premi re utilisation remplir le socle avec environ 600 ml 50 50 d thyl ne glycol et d eau afin de ne pas endommager l instrument Voir page 4 du mode d em ploi Le socle doit tre rempli m me si le refroidissement n est pas n cessaire Ne pas utiliser seulement de l eau de l anti gel commercial ou tout autre solvant organique pour remplir le socle L eau aug mente en volume lorsqu elle g le Du fait que l eau est enferm e le socle peut se fendre Les solvants organiques peuvent causer des dommages chimiques irr para bles Ne pas refroidir le socle en dessous de 20 C 4 F Refroidir le socle dans un seau rempli de glace o un dans un r frig rateur Ne pas utiliser avec un gel ou un tampon plus de 50 C Eviter le surchauffement en refroidissant le socle avant l emploi Afin d viter le surchauffement durant de longues utilisations sous haut voltage changer si possible le socle avec un sec ond socle d j refroidi Un surchauffement peut causer des dommages irr parables l instrument Si l instrument n est pas utilis en confor mit avec les recommandations du fabri quant les protections 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 Bioscien2. either by the fluorescence of bound ethidium bromide or by autoradiography 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 under a UV lamp reveals band location To check progress 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 Note Ethidium bromide slows DNA migra tion by about 15 Alternatively after electrophoresis stain the gel in an ethidium bromide solution 0 5 pg ml H O 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 light and 40 transparent to 254 nm light View the sam ple under 366 nm UV light or reduced intensity 302 nm UV light to reduce pho toknicking 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 section 6 15 Bibliography Ausubel et al eds Current Pro
3. presence of ethidium bromide Special agaroses are available that can extend resolution ranges A common standard is a Hind II digest of lambda phage which gives eight fragments ranging in size from 0 1 to 23 kb pairs For good resolution run 45 minutes on a 10 cm long 1 agarose gel in 0 5X TBE gel at 150 V Table 2 Agarose Effective range of resolution i Agarose concentrations of linear DNA fragments kb for separating 0 5 do 2 3 DNA fragments of various sizes 0 7 0 8 a 12 1 0 0 5 10 1 2 04 7 1 5 02 3 Current Protocols in Molecular Biology p 2 5 2 1993 11 12 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 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 HE 100 horizontal unit is recommended for this application rather than the HE 33 Important Do not adjust the pH of these buffers once they are prepared according to the recipe Running buffers for DNA in agarose gels Recipes for the two most commonly used running buffers for DNA electrophoresis are listed below The ionic strength of t
4. 3 97 17 Code No 80 6052 64 80 6052 45 80 6050 17 80 6052 83 80 6053 02 80 6412 31 80 6053 40 80 6053 59 80 6053 78 80 6053 97 80 6177 09 80 6053 21 80 6194 19 80 6053 40 80 6053 59 Combs Specify thickness and number of wells as tabulated below Order a comb back separately Thickness No of wells Code No mm 1 0 Preparative 80 6051 88 1 0 8 80 6051 50 1 0 12 80 6050 74 1 0 16 80 6051 12 1 5 Preparative 80 6052 07 1 5 8 80 6051 69 1 5 12 80 6050 93 1 5 16 80 6051 31 Comb back with 2 screws 80 6050 36 Replacement screws for comb backs pk 12 80 6050 55 Reagents Ethidium bromide 10 ml 17 1328 01 Agarose NA 100 g 17 0554 02 Agarose prep 50g 80 1130 07 Ficoll 400 100 g 17 0400 01 Bromophenol Blue 10g 17 1329 01 Tris 500 g 17 1321 01 EDTA disodium salt 100 g 17 1324 01 Boric acid 500g 17 1322 01 Companion products EPS 2A200 power supply 80 6406 99 EPS 301 power supply 18 1130 01 MacroVue UV 20 Transilluminator 115 V 80 6245 11 230 V 80 6245 30 Notes Notes Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences SE 751 84 Uppsala Sweden Amersham Biosciences 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Postfach 5480 D 79021 Freiburg Ficoll are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham Biosciences is a trademark of Amersham plc Tris is a tra
5. A voltage gradient of 12 V cm 150 V separates 0 1 to 23 kb fragments of a Hind III digest of DNA in 30 to 40 minutes using 1 agarose gel and 0 5X TBE running buffer Alternatively using the same solutions this sample could also be run at 24 V cm 300 V with acceptable band resolution in 20 to 30 minutes Chill the base before use Voltage Gradient Time V V cm min 500 40 5 400 31 10 300 24 20 200 16 30 to 40 150 12 30 to 60 For rapid runs of 20 minutes or less use 0 5X TBE and chill the base to 20 C before use ersham Biosciences After the separation 1 Turn off the power supply disconnect the leads and remove the lid 2 If no ethidium bromide was added to the gel or sample before the run stain the gel in a solution of 0 5 to 1 0 pg ml ethidium bromide in water or buffer 3 Clean the unit as described below Care and Maintenance Cleaning Important Never autoclave any component of the electrophoresis unit or casting kit After each use clean the unit with a mild detergent and water rinse thoroughly with distilled water and allow to air dry 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 To reduce DNase and RNase contamination soak the buffer chamber or casting kit for 10 minutes in a 3 hydrogen peroxide H O solution then rinse thoroughly with DEP
6. C treated autoclaved deionized water Sambrook et al 1 7 40 Replacing foam pads Remove worn foam pads Peel off the adhesive cover on a new foam pad Align the pad so that it will rest on the bottom of the tray along a short 7 cm side adhesive side toward the inside wall and then press it in place Repeat with second pad on the wall opposite the first pad Replacing the electrode It is recommended that electrodes be replaced only by Amersham technicians Call your local representative for advice Troubleshooting Deformed sample well Y Y Allow the gel to set for a minimum of 30 minutes and make sure it is at room temperature before removing the comb When removing the comb hold it at a slight angle and lift 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 Y Double banded pattern Y Poor band resolution Y ISISSNS SN Foam pads peel off 10 If a comb or running tray is warped replace Reduce the voltage Choose a buffer with the appropriate ionic strength and buffering capacity The buffering capacity of TBE for example is higher than that of TAE If the buffer is depleted stop the run remove the lid and pipette the buffer from each chamber into the opposite chamber to replen
7. HE 33 Mini Submarine Electrophoresis Unit User Manual 80 6273 42 Amersham BE Merci ET e Y Biosciences Mini Submarine Electrophoresis Unit Function Important Information Unpacking Specifications Operating Instructions Care and Maintenance Troubleshooting Notes Buffers and Volumes Bibliography Customer Service Information gt e 0 W N e 10 11 15 16 Figure 1 Main components See Figure 2 for an illustration of the casting kit Mini Submarine Electrophoresis Unit Function The HE 33 horizontal agarose unit is intended for rapid electrophoresis of small quantities of nucleic acids in agarose gels A gel is cast in the gel caster which holds one or two combs Eight different combs are available a maximum of 32 samples can be run if two 16 well combs are used After the gel sets the running tray is transferred to the platform of the horizontal unit The base of the unit holds coolant that can be chilled before the run This passive cooling capacity allows fast high voltage runs Color coded leads connect electrodes in the unit base to the power supply Buffer chamber Lid assembly Electrode connectors 2 Running platform supports the running tray Fill the base with 50 50 ethylene glycol water HE 33 Mini Submarine Important Information The safety lid must be in place before connecting the power leads to a power supply Turn all p
8. buffer 5X 25 Ficoll 400 0 25 Bromphenol blue 10 ml Deionized H20 to 7 0 ml Ficoll 400 2 5 9 Bromphenol blue FW 691 9 25 0 mg Deionized H20 to 10 0 ml Add to sample in proportion so that Y5 of the final volume is loading buffer Loading buffer increases solution density Notel Sucrose or glycerol may be used instead of Ficoll 400 Note2 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 comi gration with smaller nucleic acids Well volume Comb no of thickness well width sample vol per Code no wells mm mm 1 mm depth pl 80 6051 88 1 prep 2 ref 1 0 44 6 44 6 80 6052 07 1 prep 2 ref 1 5 44 6 66 9 80 6051 50 8 1 0 6 5 6 5 80 6051 69 8 1 5 6 5 9 7 80 6050 74 12 1 0 3 9 3 9 80 6050 93 12 1 5 3 9 5 8 80 6051 12 16 1 0 2 6 2 6 80 6051 31 16 1 5 2 6 3 9 The preparative combs form two reference wells for MW standards one on each side of the preparative well The first number is sample volume mm in the preparative well the second is volume mm in the reference well Caution Ethidium bromide is a known mutagen Always wear gloves when handling Wear UV safety gog gles and protect skin when using any UV light source DNA detection DNA can be detected
9. ces sont recom mand s pour l utilisation l entretien et r paration de cet appareil osciences HE 33 Mini Submarine Electrophoresis Unit 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 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 for repacking should it become necessary to Ame rsham Bi return the unit Specifications Max voltage Max wattage Max current Max operating temperature Max buffer volume Coolant required Gel size Environmental operating conditions Installation category Pollution degree Dimensions Weight base lid and leads only Product certifications 500 V for 5 minutes or less 15 W 500 mA 50 C 250 ml 600 ml 50 50 water ethylene glycol 7x10 cm Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m II 2 width x depth x height 24x13x7 cm 9 5x5 2x2 8 in 0 4 kg 0 9 Ibs EN61010 1 UL3101 1 CSA 022 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
10. demark of Union Carbide Chemicals and Plastics Co All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Amersham Biosciences 1999 All rights reserved Amersham Printed in the USA o o Biosciences
11. hese buffers is appropriate for the application do not adjust the pH of these buffers once they are prepared according to the recipe 1 10X Tris borate EDTA TBE stock buffer 0 89 M Tris 0 89 M boric acid 20 mM EDTA pH 8 2 1000 ml Tris base FW 121 1 0 89 M 108 0 g Boric acid FW 61 8 0 89 M 55 0 g EDTA solution 0 5 M pH 8 0 soln 3 0 02 M 40 0 ml 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 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 49 Acetic acid 99 5 0 20 M 11 4 ml EDTA solution 0 5 M pH 8 0 soln 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 NazEDTA 2H50 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 H20 to 100 0 ml 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 Table 3 Comb specifications 14 Sample loading buffer Loading
12. il the gel is submerged under 1 mm of solution This requires about 220 ml Load the samples Add sample to 5X sample loading buffer and mix Ys of the final volume is loading buffer see p 14 Use a micro pipette to load each sample taking care to avoid puncturing the well bottom or entrapping any bubbles Place the lid so that the cathode black lead is at the end nearest the sample well Nucleic acid samples migrate toward the anode red lead Connect the color coded leads red to red and black to black to an approved power supply such as the EPS 24200 Set the voltage level and timer if avail able according to the degree of resolution sought HE 33 Mini Note To calculate the volt age gradient divide the voltage setting by the distance between the elec trodes 12 7 cm Table 1 Voltage settings and the maximum recom mended separation time using 1 Agarose NA agarose 0 5X TBE and a chilled base Submarine Electrophoresis Unit Quick high voltage runs Certain applications such as screening samples or checking sample purity can be accomplished quickly under high voltage conditions Chill the base 20 C and limit the run to 5 minutes or less at 500 V Note At the maximum setting the unit begins overheating as soon as the chilled base reaches ambient temperature If overheating is not controlled the gel will melt and or the base of the unit will warp Slower lower voltage runs
13. ish the buffer If the gel is uneven level the casting tray before pouring the gel The comb must be vertical to prevent well shape distortion Decrease the buffer level to 1 mm above the top of the gel to reduce the vertical temperature gradient Add Ficoll glycerol or sucrose to the sample loading buffer to ensure that the sam ple sinks to the bottom of the well Ficoll is preferred Make sure the sample is completely dissolved Reduce the voltage Reduce the sample concentration Reduce the sample volume At least 1 mm of gel below the bottom of the comb is required to prevent sam ples from leaking out of the well 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 Install the running tray as as described on page 4 do not press straight down into place 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 2 below Other factors affecting separation results include the running buffer voltage setting temperature conformation and the
14. n hour before use The base will always be ready if you store it in the refrigerator or freezer Caution Ethidium bromide is a known mutagen Always wear gloves when handling Figure 2 Gel casting kit Approach the foam pad with one end of the running tray 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 before sealing against the foam pad Prepare solutions 1 Prepare 250 ml of running buffer Refer to p 13 for recipes of commonly used electrophoretic running buffers Prepare the sample loading buffer Refer to p 14 for a recipe and a table of volume requirements for each comb size Prepare approximately 7 ml agarose solution per mm of gel thickness For example a 3 mm gel requires 0 3 cm x 7 cm x 10 cm 21 ml Dissolve agarose in running buffer heat according to instructions accompa nying the agarose and allow the solution to cool to 50 C before pouring into the casting tray Optional Add 0 5 pg ml ethidium bromide to the gel solution to observe sep aration during electrophoresis Cast the gel 1 Foam pads 2 Install the running tray Firmly grasp the casting tray with one hand With the other hand place one end of the running tray against the foam pad at the bot tom edge press the tray against the pad and then lower it to rest on the bot tom of the casting tray seating the o
15. ne 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 Remove the running tray and gel by grasping the handles of the tray and press ing against one of the foam pads Once the tray clears the opposite pad lift it out Transfer the running tray and gel to the chilled base Caution Ethidium bromide is a known mutagen Always wear gloves when handling Wear UV safety gog gles and protect skin when using a UV lamp Note If no dye was added to the coolant place the base on a dark background to see the wells more easily The electrophoresis run Refer to the Notes Buffers and Volumes section for additional information and guidelines 1 4 Chill the base before use especially when higher voltage settings will be used or when the separation will require more than 30 minutes Note To monitor separation progress either add 0 5 pg ml final conc of ethidium bromide to the running buffer now or add 50 pg 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 Adding ethidium bromide to the running or sample buffer slows migration slightly Detection by this method is not as sensitive as staining and viewing on a transilluminator See DNA detection p 15 Fill both buffer chambers with running buffer unt
16. or products recommended or approved by Amersham Biosciences osc i enc es 3 Important Do not fill the base with commercial antifreeze organic solvents or pure water Note It is not necessary to replace the coolant Operating Instructions Agarose gels are first prepared using the gel casting kit Samples are then loaded into wells and electrophoretically separated The fluorescent dye ethidium bromide can be added to the gel or electrophoresis buffer or both to track separation progress After electrophoresis the gel may be stained and photographed blotted or dried for autoradiography Fill the base with coolant Even if no cooling is required it is important to fill the base with the proper coolant solution before the first use because the solution provides a necessary heat sink 1 Prepare 600 ml of 50 50 ethylene glycol water Optional To help see wells better while loading the sample add a drop or two of soluble dye or food color to the coolant solution Locate the inlet hole on the bottom of the base near the edge Fill the base cavity as full as possible with coolant using a 50 ml syringe or pump 2 Fit the threaded white plastic plug into the hole and taking care that the thread is properly engaged screw it in place until it is snug Tighten another Ys to Ya turn to prevent leaks 3 Place the prepared base in an ice bucket or into a refrigerator or freezer set no lower than 20 C for about a
17. ower supply controls off and disconnect the power leads before removing the safety lid A Electrophoresis Unit Informations importantes D 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 cu rit Before the first use fill the base with about 600 ml 50 50 ethylene gly col water to prevent irreparable dam age to the unit See instructions on page 4 The base must be filled even if no cooling is required Do not use pure water commercial anti freeze or any organic solvent to fill the base Water expands as it freezes Because the water is trapped in the base it may crack Organic sol vents will cause irreparable chemical damage to the unit Do not chill the base below 20 C 4 F Chill the base in a bucket of ice refrigerator or in a freezer Do not operate with gel or buffer tem perature above 50 C Prevent overheating by chilling the base prior to use To prevent overheat ing during prolonged high voltage runs exchange the first base with an extra prechilled second base if one is available Overheating will cause irreparable damage to the unit If this equipment is used in a manner not specified by the manufacturer the protection provided by the equip ment may be impaired Only accessories and parts approved or supplied by Amersham Biosciences may
18. ther end of the tray against the opposite foam pad UV transparent running tray Cast the gel on this tray then transfer gel to the horizontal unit base for electrophoresis Gel casting tray Figure 3 A comb back which fits onto the rim of the casting tray posi tions the comb in the gel Two screws hold the adjustable comb For twice as many wells a second comb can be placed in the center of the gel Prepare the comb s Fit the two slots in the comb between the loosened thumb screw heads and the comb back Tighten the screws until the comb is just supported Seat the comb assembly on the rim of the casting tray and adjust the bottom of the comb so that it is about 1 0 mm from the running tray Tighten the screws to secure the comb To run twice as many samples prepare two combs N Comb back Screws 2 Comb Remove the comb assembly Place the casting assembly on a leveling surface and level using the spirit level on the running tray as a guide Pour the agarose solution cooled to 50 C into the casting tray Orient the comb assembly so that the comb faces the nearest foam pad and seat it on the tray rim Check that the comb is vertical to prevent well shape distortions To run twice as many samples place the second comb assembly in the center of the tray Allow a minimum of 30 minutes for the gel to set Once the gel is set remove the comb carefully Partially lift and slightly tilt the comb at o
19. tocols in Molecular Biology Greene Publishing and Wiley Interscience New York 1993 Sambrook J Fritsch E F and Maniatis T Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press 1989 15 16 Customer Service Information Technical Service and Repair Amersham Biosciences offers complete technical support for all our prod ucts 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 sales office or representative Important Request acopy ofthe Amersham Health and Safety Declaration Form before returning the item No items can be accepted for servic ing or return unless this form is properly completed Ordering Information All quantities are 1 except where indicated Basic unit and kit Mini Submarine Electrophoresis Unit basic Includes gel running tray gel casting tray and bubble level Order comb and comb back separately Mini Submarine Electrophoresis Unit kit Same as above plus one 8 well 1 5 mm thick comb comb back and screws Replacement parts Buffer chamber assembly Lid with power cables Fill plug bottom Fill plug top Gel running tray UVT 7x10 cm Casting tray 7x10 cm Gel casting kit with gel casting tray and running tray 7x10 cm Foam gaskets pk 2 High voltage leads Electrode replacement kit Bubble level 80 6177 09 80 6052 83 80 6050 17 80 605
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