D2 and D3-14 Horizonal Electrophoresis Systems User Manual
Contents
1. D2 RL 25D Rapid Load Rapid Load Rapid Load 1 10 1 10 1 20 1 20 1 40 1 40 1 M 1 M 2 R 2 R 2 RL RL C D C D C D TC TD Rapid Load No of Teeth N 0 N pan oa o o 0 30 ah 30 2x on o on o on o on o on o on 0 0 15 1x 15 1X 9 1X 9 1X 25 2X 25 2X Thickness Width of a of Teeth Recommended Loading Volumes PJ sl 1 0 5 2 4 14 23 33 gt N CO o NI NI i 99 NI ep a N w a ga oO Ar N 1 Loading Volume is calculated as 75 of total well volume TxWxHx0 75 2 Gel Thickness 3 8 amp 12 Channel Pipette Format 8 2 Horizontal Electrophoresis System Thermo Scientific Section 8 Optional Equipment Table 8 2 Comb Options for Model D3 14 Thickness Width of Catalog No Comb Type No of Teeth Recommended Loading Volumes PE a E CA EC fo CC CC CC CA CC fo O LC po lu loco l5 hh pp pm lo htt COMINO mm ll 1 Loading Volume is calculated as 75 of total well volume TxWxHx0 75 2 Gel Thickness 3 8 amp 12 Channel Pipette Format Thermo Scientific Horizontal Electrophoresis System 8 3 Section 8 Optional Equipment 8 4 How to Determine Well Sample Volume Horizontal Electrophoresis System Hg height of gel used Hs height of well used for sample volume Hw well height There are two volumes to consider when determining the sample volume for a horizontal gel Gel Volume which is Wid2. enoldde Jod anib snu zu wyed q sadinlag esiuyos ay poled jueJiem jeuibiio y puoAaq Jed jueuodulos ay 0 Jo Juswd nba ay JOULE O jueJem HU puajxe zou eys jueJem siy Jopun Juswd nba Jo sped Jusuodwos jo Aleda JO juswase day A UB1JEM SIU wo papn oxa sue sjoxseb pue sa ssejb susy jqepu dxa suredes Aue jo aeoueuuo J9d 0 JOUd uopOe1p pue uopeuiwazap AE UE Jo pajoejuo9 aq SNW Juswpedag samas peoiluyos oul juswadibe Ajueem siy Aq pala nod jou SI uoge pue uo e4q es uonejje suj oqe Bundaoxa esuadxa s OWJay je paveda 84 WA dIYSUBWYIOM JO ellajew UI Bulwjojuoo uou aq 0 UBAOId sped Jusuoduos syjuow 9 is Ay 3814 ey Buung Jaumo juanbasqns Aue o spuajxa uo 98 01d AJUBLIEM ay palenipep si juauudinba noA w ewes ay jejeuixoJdde je joaya OU OB Wu AIUEUEA ay os awin Buiddiys smojje s y AuIEL Ino wo paddiys si juswdinba Ino A app ay wo syjuow OM SUEIS polad AIUEUEAMA SUL TIWNOILYNYSLNI ALNVYYVM SLINAOYd IMO SISILNSIDS 43HSIA OWYIHL Thermo Scientific Horizontal Electrophoresis System 9 2 Thermo Fisher Scientific 401 Millcreek Road Marietta Ohio 45750 United States www thermofisher com ThermoFisher SCIENTIFIC
3. or an agarose additive may be added to standard or low melting point agarose Example A good mid range gel percentage would be 0 7 or 0 7g agarose in 100ml electrophoresis buffer TBE or TAE following heating and dissolving the agarose 10ul of ethidium bromide stock solution Smg ml is added The gel would be run with compatible electrophoretic running buffer 1X TBE or 1X TAE that also contained ethidium bromide One liter of the running buffer would contain 100ul of this 5mg ml ethidium bromide stock solution 2 Ethidium Bromide For photodocumentation of samples the gel may be stained during or following the run with a variety of stains The most common stain for DNA is ethidium bromide Ethidium bromide may be added directly to the gel and running buffer to visualize and photograph the separated fragments following the gel run without the need for an additonal staining step The ethidium bromide is added to both the gel after heating and the electrophoresis buffer at a concentration of 0 5ug ml Conversely the gel may be stained in a concentrated ethidium bromide solution after the gel run and rinsed for visualization Warning Ethidium bromide is a potential carcinogen Care in handling the powder and stock solution must be taken Always wear gloves when handling the powder solutions and all gels that contain ethidium bromide Thermo Scientific Thermo Scientific Section 5 Reagents Information Table 5 3 Preparatio
4. 4 liter bottle Rnase AWAY is a registered trademark of Molecular BioProducts Horizontal Electrophoresis System 4 1 Thermo Scientific Section 5 Reagents Information Selection of Reagents for Gel Electrophoresis 1 Agarose There are various types of agarose commercially available that may be used Besides standard ultra pure electrophoresis grade agarose there are also numerous low melting point products for easy sample recovery as well as specialty products formulated for specific uses i e to separate and or recover very small or very large fragments Table 5 1 Mobility range of DNA in different percentage agarose gels Approximate range of 0 SUE separated DNA fragments kb 30 to 1 Be Table 5 2 Amount of Agarose to prepare Gel volume is determined by the following formula and may be adjusted according to need or preference gel width cm X gel length cm X gel thickness cm ml of agarose D3 14 23 x14 80 5ml 161ml 241 5ml 322ml 14 4 x 10 2 36 72 73 44 110 16 146 88 Horizontal Electrophoresis System 5 1 Section 5 Reagents Information 5 2 Horizontal Electrophoresis System Selection of Reagents for Gel Electrophoresis continued Note An increased agarose provides better separation of small fragments and bands very close together that tend to be more difficult to separate A specialty agarose product formulated to increase resolution of low molecular mass samples may also be used
5. Technical Services to schedule a repair Samples that appear to run backwards through the gel is caused by the tray being placed in the chamber in the reverse direction The tray should be placed in the chamber with the comb at the edge of the tray closest to the cathode side of the chamber Samples are not moving as expected through the gel remaining in the wells running backwards or diffusing into the gel Thermo Scientific Horizontal Electrophoresis System 6 1 Section 6 Troubleshooting Problem Always make sure to allow the gel to solidify completely before moving the tray When the comb is removed from the gel the sample well unit or removing the comb To avoid damage to the sample wells gently rock the is ripped and damaged comb back and forth lightly to loosen then slowly pull the comb straight up out of the gel tray This rocking helps to avoid suction as the comb is removed The volume of running buffer used to submerge the gel should only be between 3 5mm over the gel surface Thw gel should be completely submerged to avoid the gel from drying out which can smear the bands and possibly melt the gel due to overheating If excessive running buffer is added the mobility of the DNA decreases and band distortion may result Excess buffer causes heat to build up and buffer condensation inside the unit may result The gel seems to run slower under usual running conditions Additional Sources for Reference Mania
6. Horizontal Electrophoresis System Model D2 and D3 14 Operating and Maintenance Manual 7007320 Rev 1 Visit us online to register your warranty www thermoscientific com warranty Gi Owl Preface Model D3 14 Horizontal Gel System Gel System Model D2 Horizontal MANUAL NUMBER 7007320 1 28432 SI 11500 9 10 12 Added lid assembly part number to pg 1 2 parts list ccs 0 5 29 12 Transferr to Marietta was Rev Date 3 2005 CCS REV ECR ECN DATE DESCRIPTION By Thermo Scientific Horizontal Electrophoresis System i Preface CAUTION Contains Parts and Assemblies Susceptible to Damage by Flectrostatic Discharge ESD Important Read this instruction manual Failure to read understand and follow the instructions in this manual may result in damage to the unit injury to operating personnel and poor equipment performance A Caution All internal adjustments and maintenance must be performed by qualified service personnel A Warning To avoid the risk of personal shock always disconnect the gel box from the power supply Further the power supply must be equipped with a shut down on disconnect circuit Do not move the unit unless the power source to the unit has been disconnected Running conditions for this unit should not exceed the name plate readings found on the lower buffer chamber NEVER leave unit running unattended A Statement of Proper Use Use this product only for its intended purpose as described in t
7. attached to the back of the unit at the junction of the lids attached power cords to the banana plugs 2 For shipping and convenient storage the gasketed gel tray is packaged inside the external gel caster upon arrival To remove the gel tray lift it out of the caster by placing both hands firmly on the shorter tray ends and pull up slowly from the caster at an angle Tray needs to fit snug for leakproof gel casting so it may be tight Walking the tray upward at an angle may be helpful 3 To cast gels place the gel tray into the external gel caster making sure the tray is pushed all the way down and level in the external gel caster For best results be sure to cast on a level surface Leveling platforms are available through Technical Services Catalog No B LP pala Figure 2 1 Model D2 Figure 2 2 Model D3 14 4 Casting a Level Gel The need to cast a level gel is very important for consistent reproducible results Level the caster by placing the enclosed bubble level Model D3 14 only order B LP for Model D2 in the center of the gel tray Using the thumbscrews on each side of the caster slowly turning one thumbscrew front only at a time and lining up the bubble in the level with the center circle Check various areas on the gel tray by moving the bubble level to each end of the gel tray to ensure you have leveled correctly Note It is wise to always run a sample lane of a known standard ladder to determine
8. concentration and size of separated fragments after the gel run and to aid in photo documentation and analysis A Thermo Scientific Horizontal Electrophoresis System 2 1 Section 2 Casting a Gel 2 2 Horizontal Electrophoresis System 5 Preparing the Gel Using electrophoresis grade agarose and compatible electrophoresis buffer the gel may be prepared in various ways The percentage of agarose and the electrophoretic buffer used is determined by the size of the samples to be separated and further recovery of the samples The agarose and buffer are mixed and heated using a heat source in a microwave oven or in an autoclave until the agarose is completely dissolved The prepared gel then must be cooled to below 60 C before casting to avoid warping the UVT gel tray due to excessive heat If numerous gels are to be run in one day a large volume of gel may be prepared and placed in a covered bottle stored between 40 60 C in a water bath This provides a ready gel supply in a warm liquid form that will solidify quickly when gels are cast 6 Pour or pipette the correct amount of warm agarose lt 60 C onto the UVT gel tray Immediately after pouring insert the desired comb or combs into the comb slots to form the sample wells If only a small portion of gel is required for proper sample separation up to 4 combs may be used to run 2 3 or 4 sets of equal distance samples simultaneously expanding the number of samples per gel that may b
9. e run Note A higher percentage of agarose gt 0 5 may give the best results when using 4 of the 50 tooth D3 14 only D2 goes to 40 combs to avoid damage to the wells when combs are removed A el Figure 2 2 Model D3 14 Process Thermo Scientific Thermo Scientific Section 2 Casting a Gel Wall Comb To conserve agarose a wall comb may also be used to divide and use a shorter length of the gel tray If a wall comb is used pipette a bead of agarose along the bottom and side edges of the wall comb once it has been placed in the tray to seal the combs edges to the trays bottom and sides Once this bead is solidified the cooled gel may be poured as described Alternately regular tape cut slightly longer then the comb can be placed flat along the comb s surface and the comb angled into place in the gel tray Extra tape is then placed on the outside of the comb in the excess tray area to reinforce the corners Allow the gel to solidify completely Standard agarose should solidify completely in about 30 minutes If low melting point or a specialty agarose is used consult the instructions that came with the product Casting Dam A casting dam may also A be used The dam blocks off a portion ra of the UVT gel tray to allow shorter gels to be cast and run in one tray Because it is manufactured using high quality aluminum that seals the agarose upon contact there is no need for tape The Casting Dam is free standing so the ch
10. er percentage of agarose 50 596 may be wise to avoid damage to the sample wells Low percentage gel and the small sample wells may cause the sides of the wells to collapse when the comb is removed A higher percentage of agarose forms a tighter gel matrix Casting a slightly thicker gel may also remedy this problem Note Combs may also be removed prior to pouring buffer in buffer chamber A 4 Load prepared samples into the wells Samples should be mixed with a sample loading buffer giving weight to the samples so that they drop evenly into the wells and contain tracking dyes to monitor the gel run Refer to page 16 The combs supplied with the D3 14 unit are designed in a micro well format This format allows speedy sample loading using a multi channel pipette The 25 tooth comb is in the 1X micro well format and matches each tip of the multichannel pipette while the 50 tooth comb is in the 2X format loading every other lane The D2 has a 1X 15 well comb and a 2X 30 well comb Horizontal Electrophoresis System 3 1 Section 3 Using the System 3 2 Running a Gel 5 Carefully slide the SuperSafe lid with attached power supply leads continued onto the unit This will connect the power supply leads to the banana plug electrodes and complete the circuit Plug the other end of the power supply leads into appropriate power supply red to red black to black 6 Turn on the power supply See Recommended Running Conditions Ca
11. er each use including the UVT gel tray Rinsing the UVT gel tray will avoid any salt build up in the gasket material from the electrophoretic running buffer extending the gasket life and ensuring leak free gel casting Thermo Scientific Horizontal Electrophoresis System 3 3 Thermo Scientific Section4 Care and Cleaning Caution Do not use ethanol or other organic solvents to clean these products Organic solvents cause acrylic to craze or crack Clean all acrylic systems with warm water and a mild detergent Do not autoclave bake or microwave your unit Temperatures over 50 C can do damage to the acrylic A The unit may be rinsed with warm water or cleaned with warm water and a mild detergent to get rid of any debris Note If an RNase free electrophoresis system is desired there are various methods to rid the system of RNA contamination For fast and easy decontamination use RNase AWAY Spray wipe or soak labware with RNase Away then wipe or rinse the surface clean it instantly eliminates RNase RNase Away eliminates the old methods that include treatment with 0 1 Diethyl Pyrocarbonate DEPC treated water and soaking in dilute bleach DEPC is suspected to be a carcinogen and should be handled with care This electrophoresis system should never be autoclaved baked or placed in a microwave To order RNase AWAY contact Technical Services 7000 250ml bottle 7002 475ml spray bottle 7003 1 liter bottle 7005
12. his manual Do not use this product if the power leads are damaged or if any of its surfaces are cracked Material in this manual is for information purposes only The contents and the product it describes are subject to change without notice Thermo Fisher Scientific makes no representations or warranties with respect to this manual In no event shall Thermo be held liable for any damages direct or incidental arising out of or related to the use of this manual 2012 Thermo Fisher Scientific All rights reserved Horizontal Electrophoresis System Thermo Scientific Preface Important operating and or maintenance instructions Read the accompanying text carefully gt gt gt Potential electrical hazards Only qualified persons should perform procedures associated with this symbol re gt Equipment being maintained or serviced must be turned off and locked off to prevent possible injury Hot surface s present which may cause burns to unprotected skin or to materials which may be damaged by elevated temperatures Marking of electrical and electronic equipment which applies to electrical and electronic equipment falling under the Directive 2002 96 EC WEEE and the equipment that has been put on the market after 13 August 2005 Lars This product is required to comply with the European Union s Waste Electrical amp Electronic Equipment WEEE Directive 2002 96 EC It is marked with the WEEE symbol Thermo Fishe
13. l D2 Wide Electrophoresis System 1 2 Horizontal Electrophoresis System Gel Size o oo o o 14 4cm W x 10 2cm L Footprint s s s 17cm W x 17cm Lx 10cm H Running Buffer Volume 44 AN SE SES E 600ml COMPLETE SYSTEM INCLUDES e Buffer Chamber e SuperSafe Lid with Attached Power Supply Leads EasyCast Gasketed U V Transmissible UVT Gel Tray e External Multiple Gel Caster e 4 Combs 2 30 Well and 2 40 Well Power Supply Super Safe Leads Lid NG External Multiple Gel Caster UVT Gel Gasket 2 Hay Buffer Chamber Complete System with Buffer Exchange Ports D2 BP Thermo Scientific Section 1 Introduction Model D3 14 Wide Gel Size 6 dieu acne 6d wus dead ave 23cm W x 14cm L Electrophoresis System Footprint 26cm W x 29cm L x 8cm H Running Buffer Volume 800ml COMPLETE SYSTEM INCLUDES Buffer Chamber SuperSafe Lid with Attached Power Supply Leads EasyCast Gasketed U V Transmissible UVT Gel Tray External Gel Caster 4 Combs 30 Well Power Suppl Super Safe External Gel Gasket 2 SA Caster y Buffer Chamber Bubble Level O Comb 4 Thermo Scientific Horizontal Electrophoresis System 1 3 Section 3 Casting a Gel 1 Remove the SuperSafe lid from the gel box by holding the front of the buffer chamber with one hand and pulling the lid off sliding off evenly by holding the center of the back of the lid The cover is
14. n and Properties of TAE and TBE Electrophoresis Buffer Systems These buffers are used because they both have a basic pH which gives the phosphate group of the DNA a net negative charge allowing migration of the DNA toward the positive anode in the electrophoresis chamber TAE Tris Acetate with EDTA 40mM Tris Base 40mM Acetic Acid 1mM EDTA ETE AN ES TBE Tris Borate with EDTA 89mM Tris Base 89mM Boric Acid 2mM EDTA CI Pen I Horizontal Electrophoresis System 5 3 Section 5 Reagents Information 5 4 Horizontal Electrophoresis System Choose the buffer best suited to the experiment Each buffer has different properties providing the necessary ions for electophoretic migration Buffer Suggested Use TAE Buffer e Use when DNA is to be recovered e For electrophoresis of large 520kb DNA e Applications requiring high resolution e Has low ionic strength and low buffering capacity recirculation may be necessary for long runs 54hrs TBE Buffer e General Purpose Buffer e Can be re used e For electrophoresis of small lt 1kb DNA e Better resolution of small lt 1kb DNA e Decreased DNA mobility e High ionic strength and high buffering capacity recirculation may not be required for extended run times e Reacts with the agarose making smaller pores and a tighter matrix This reduces broadening of the DNA bands for sharper resolution 3 Sample Buffer Samples are prepared and mixed with sample buffer before bei
15. nb si a91nas judwdinboa y as9ueuajulew saund pue uvonejado uone e sui Juawd nba lejap AjjnjaJeo sjenuew UO ONSU pajulid SOALIE juaudinba JnoA 810599 uongwJoju uoneJedald aus aaisuayalduwoso YIM djay 0 Apea s 891yO sajes OWE 890 INOA s onpoJld JO sso JO SyyOuJd sot 0 saBewep uonejiwi noyym Hulpnjoul sebewep enuenbasuos Jo Pal pu Aue JO a qe aq zou eys oway AlddV TIVHS 3ASOdYNd YVINOIILYYd V 404 SSINLIA YO ALIAVLNYHOIYAN JO SILLNVIYVM ON ANNAN YO IVYO NFLLIUM YIHLIHM SSILNVYYVM Y3HLO TIY 40 NAIN NI ANY FAISNTOX3 SI ALNVYYVM SIHL uoneunsep gO peddiys ose syed juawsoe dal pue pied abe sod oway 0 pauinjai aq Isnw syed BulWlojuoo uou e uondo sowJay 1y Tuswdinba Jo jusuodwoo Aue jo unya Jo eAOJdde Jod anib snw juauyuedag sexinas EIIUY99L ay poled uenem eui6iJo ay puoAaq Jed juduodwWoo ay 0 Jo juawdinba ay Jayya O jueJem ay puajxa JOU eys jueJem siy apun Juswd nba Jo sped jusuodwoo jo edau Jo juawae day AJUCIIEM SIY wo papn oxa ase sjayseb pue sia ssejB sway ajgepuadxy sed Aue jo adue woad 0 JOLId UONOSJIP pue UONEUIWISISP jueJJem 104 pajoejuo9 aq snw juauyledag sexies esiuyoa ay juswaaibe AjueJem siy Aq pala nod zou SI uoge pue uopesqyeo uonejesuj Joge Buipnpul esuadxa s owJey ye pasejda aq IIM d ysuewy Jom JO enayeu Ul Bulwjojuoo uou aq 0 UBAOJd syed juauoduloo syjuow oc xis AJy 38114 ayy Bung Jaumo juanbasqns Aue o spuaj
16. ng applied to the prepared gel Sample buffers contain similar components to the running buffer dyes for visibility and glycerol to provide weight to the samples This increased sample density ensures samples load evenly into the wells and do not float out during loading Dyes also migrate toward the anode end of the electrophoresis chamber at predictable rates allowing the gel run to be monitored 4 DNA Markers Markers are run on each gel to monitor sample separation and to provide an accurate size estimation of the samples By running a known marker of a specific concentration the amount of the DNA can be estimated These size markers are a suitable restriction digest of commonly available DNA Thermo Scientific Section6 Troubleshooting Check to see if the gasket is firmly seated in the grooves on the ends of the UVT Agarose leaks into chamber when pouring gel gel tray Reseat gasket if necessary by removing and rinsing under warm running water then reseat evenly in the tray groove Check to be sure the casting is being done on a level surface A leveling platform may be required Make sure the gel tray is pressed all the way down and rests Bands seem to be running at an angle level on the casting chamber platform the bubble in the bubble level should rest in the center circle Adjust the leveling screws to make the casting chamber D4 CST level Check to be sure the platinum electrode wire is intact and running evenly across
17. ontal Electrophoresis System Thermo Scientific Section 3 Using the System Loading the Sample d Load prepared samples into the wells Samples should be mixed in Gel co ntinue d with a sample loading buffer giving weight to the samples so that they drop evenly into the wells and contain tracking dye to monitor the gel run See Table 5 2 in Section 5 for approximate well volumes Note To run one gel follow steps a b c and d It is recommended to always run a sample lane of a known 23 130 standard ladder or marker to determine concentration and size of separated fragments after the gel run and to aid 79 416 in photodocumentation and analysis Migration patterns 6 557 and fragment sizes for commonly used DNA molecular weight markers are shown here 4 361 2 322 2 027 Finish 1 When the gel run is complete and tracking dye has migrated as far through the gel as desired or to the end of the gel turn off the power supply and slide off the SuperSafe lid to disconnect from the power source Carefully remove the UVT gel tray containing the gel wear gloves if ethidium bromide is present The UV transparent gel tray makes visualization and photography with a UV light source easy without the need to remove the gel from the tray The UVT gel tray may be placed back into the casting chamber for convenient transport to the darkroom to avoid damage to the gel 2 The gel box should be rinsed under warm running water aft
18. osen length is not restricted Catalog No DAM 23 d Pa Figure 2 3 Casting Dam Horizontal Electrophoresis System 2 3 Thermo Scientific Running a Gel Section3 Using the System l Once the gel is completely solidified carefully lift the tray s out of the external gel caster and place into the buffer chamber The running position of the tray exposes the open ends of the agarose to the buffer Pour enough compatible running buffer into the unit to fill both ends of buffer chamber and completely cover and submerge the gel Correct buffer level is clearly marked on the side wall as FILL LINE See Recommended Running Conditions for approximate buffer volumes needed for your unit Too little buffer may cause the gel to dry out during the run while excess buffer may decrease DNA mobility and cause band distortion Carefully remove the comb or combs by tapping lightly to loosen and slowly lifting straight up out of the gel tray To avoid damage to the sample wells always make sure to allow the gel to solidify completely before moving the buffer chamber gel tray or removing the combs After placing the gel tray into the unit in the running position submerge the gel in 3 5mm of running buffer Lightly tap each comb gently back and forth to loosen then slowly pull the comb straight up out of the gel tray This will break any suction that may exist between the gel and comb When using all four combs D3 14 model a high
19. r Scientific has contracted with one or more recycling disposal companies in each EU Member State European Country and this product should be disposed of or recycled through them Further information on Thermo s compliance with this directive the recyclers in your country and information on Thermo products will be available at www thermofisher com Y Always use the proper protective equipment clothing gloves goggles etc Y Always dissipate extreme cold or heat and wear protective clothing Y Always follow good hygiene practices Y Each individual is responsible for his or her own safety Thermo Scientific Horizontal Electrophoresis System lil Preface Do You Need Information or Assistance on Thermo Scientific Products If you do please contact us 8 00 a m to 6 00 p m Eastern Time at 1 740 373 4763 Direct 1 800 438 4851 Toll Free U S and Canada 1 877 213 8051 FAX http www thermoscientific com Internet Worldwide Web Home Page service led marietta thermofisher com Tech Support Email Address www unitylabservices com Certified Service Web Page Our Sales Support staff can provide information on pricing and give you quotations We can take your order and provide delivery information on major equipment items or make arrangements to have your local sales representative contact you Our products are listed on the Internet and we can be contacted through our Internet home page Our Service Support staff can supply technical info
20. refully monitor the gel run to avoid samples running into the path of another set of samples Loading the Sample in Gel There are two ways to load the gel s Dry Loading and Wet Loading DRY LOADING loading the sample in the gel without the presence of buffer a Remove the gel tray from the casting chamber b Load the sample into the gel but be careful not to puncture the bottom of the gel Place the gel tray into the buffer chamber in the running position c Load the sample into the gel but be careful not to puncture the bottom of the gel Place the gel tray into the buffer chamber in the running position d Carefully fill the buffer chamber with buffer to cover UVT tray fill line completely cover and submerge the gel See Recommended Running Conditions for approximate buffer volumes needed for your unit Too little buffer may cause the gel to dry out during the run while excess buffer may slow DNA migration in the gel WET LOADING loading the sample in the gel when it is submerged in buffer a Remove the gel tray from the casting chamber b Place the gel tray into the buffer chamber in the running position c Pour running buffer into the unit to fill chamber and completely cover and submerge the gel See Recommended Running Conditions for approximate buffer volumes needed for your unit Too little buffer may cause the gel to dry out during the run while excess buffer may slow DNA migration in the gel Horiz
21. rmation about proper setup operation or troubleshooting of your equipment We can fill your needs for spare or replacement parts or provide you with on site service We can also provide you with a quotation on our Extended Warranty for your Thermo Scientific products Whatever Thermo Scientific products you need or use we will be happy to discuss your applications If you are experiencing technical problems working together we will help you locate the problem and chances are correct it yourself over the telephone without a service call When more extensive service is necessary we will assist you with direct factory trained technicians or a qualified service organization for on the spot repair If your service need is covered by the warranty we will arrange for the unit to be repaired at our expense and to your satisfaction Regardless of your needs our professional telephone technicians are available to assist you Monday through Friday from 8 00 a m to 6 00 p m Eastern Time Please contact us by telephone or fax If you wish to write our mailing address is Thermo Fisher Scientific 401 Millcreek Road Box 649 Marietta OH 45750 International customers please contact your local Thermo Scientific distributor v Horizontal Electrophoresis System Thermo Scientific Thermo Scientific Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Section 8 Section 9 Table of Contents Introd
22. th x Length x Gel Height and uses centimeters Sample Volume which is Tooth Width x Comb Thickness x Apparent Well Height and uses millimeters Gel Height is generally set to a height between 0 5 cm and 0 75 cm Therefore once you choose the height the volume is the gel dimensions given in the catalog for each gel box I D times this height Gel Tray Side Hs Loading Volume Gel Tray Bottom Space Figure 8 1 Determining Volume Once the gel height Hg is chosen the well volume and then the sample volume can be calculated The well height Hw is 1 5 mm less then the gel height Hw Gel Height 1 5 mm Using the well height the volume of the well is calculated Vw Well Height Tooth width x comb thickness The loading volume is a 0 75 safety factor applied to the well volume Vs Vw 75 For Owl Combs there are only two thicknesses 1 0mm and 1 5mm This is the depth The width of the well is determined by the number of teeth For a given gel box as the number of teeth increase the volume of each tooth decreases Thermo Scientific Section 9 Warranty Information RECETTE 1006 OSI CHG 0 Noy UONEWJOJU A uUBJEM 10 JOJNQUISIP E20 IN0A j9ejuos YSN 24 apisimo suonesidde elo ads pue sonas soueuajulew uonelado Aueem juswdinba uo suonsanb noA Jamsue o peal 919M 29 8 8 08 L Jo epeueo pue VSN 1584 864 008 L Je Juswpedag souge EIIUY99L InoA peo aseajd p
23. the base of the chamber and up the side to the junction of the banana plug H there appears to be a break in the electrode connection contact Technical Services Samples seem to be running unevenly in certain areas immediately This problem may also be caused by regular casting with very hot agarose gel gt 60 F which may damage the gel tray over time Always cool the melted agarose to below 60 F before casting to avoid warping the UVT gel tray Warping the gel tray will cause all subsequent gels to be cast unevenly Gels should be no more than 5mm thick and allowed to solidify completely before running For standard agarose this would be about 30 minutes if low melting point agarose is used it may be necessary to completely solidify gels at a cooler temperature in the refrigerator or cold room Gels should be submerged in 3 5mm of buffer to avoid gel dry out but excess buffer gt 5mm can cause decreased DNA mobility and band distortion Samples do not band sharply and appear diffuse in the gel Check to be sure that a complete power circuit is achieved between the unit and the power supply Platinum wire and banana plugs should be intact To test sim ply fill the unit with running buffer and attach to the power supply without a gel or gel tray in the unit The platinum wires on both sides of the unit should produce small bubbles as the current passes through If a complete circuit does not exist there will be little to no bubbles Contact
24. tis T E E Fritsch and J Sambrook Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY Short Protocols in Molecular Biology A Compendium of Methods from Current Protocols in Molecular Biology Edited by Fredrick M Ausubel et al Adams D and R Ogden Electrophoresis in Agarose and Acrylamide Gels Methods in Enzymology Vol 152 1987 Academic Press Inc Fotador U Simultaneous Use of Standard and Low Melting Agarose for the Separation and Isolation of DNA by Electrophoresis Bio Techniques Vol 10 No 2 1991 Boots S Gel Electrophoresis of DNA Analytical Chemistry Vol 61 No 8 April 15 1989 6 2 Horizontal Electrophoresis System Thermo Scientific Thermo Scientific Section7 Specifications Recommended Running Conditions Model Leet ae a Ee eh Ma KC ana D3 14 D2 Gel Size W x Lin cm era 23x14 14 4 x 10 2 Buffer Capacity ml sp ENEE 800ml 600ml Voltage Requirements V 20 150 20 150 Time Requirements min 24s i265 rad 30 60 30 60 Migration Distance The charts below give the migration distance for each comb slot on the gel tray with the run lengths Figure 7 1 D3 14 Migration Distance Figure 7 2 D2 Migration Distance Y y One comb for a 12 8cm run length One comb for a 92cm run length Two combs for two 4 4cm run Four combs for four 3cm run lenaths Horizontal Electrophoresis System 7 1 Section8 Optional Equipment Buffer E
25. uction 00 a rd Ae 1 1 Model D2 Wide Electrophoresis System 1 2 Model D3 14 Wide Electrophoresis System 1 3 Casting a CT EE 2 1 Using the System 3 1 R nning a e EE 3 1 Loading the Sample in Gel Jc cies eo ars ei KUDA 3 2 Finish A eben kee cath de BA G ae 3 3 Care and Cleaning 4a 4 1 Reagents Information 5 1 Selection of Reagents for Gel Electrophoresis 5 1 Troubleshooting nes se lowdehwstente ncaa ews 6 1 Specifications 20 ii A 7 1 Optional Equipment nennen eee nenn 8 1 Butter Exchange Port sni vette ada nd Bee ana 8 1 A O kaa deht Panda Wario fh ate aoe 42 eu 8 2 How to Determine Well Sample Volume 8 4 Warranty Information 9 1 Horizontal Electrophoresis System V Thermo Scientific Section1 Introduction The Owl Model D3 14 and Model D2 Horizontal Agarose Gel Electrophoresis Systems are designed to provide flat even banding patterns and consistent results with hassle free gel casting No tape grease agarose seals or other accessories are required A stand alone casting platform is included for casting 1 or 2 D2 only gels simultaneously Custom combs are available upon request BEFORE STARTING Unpack the unit and inventory your order If any parts are missing contact Technical Services within 48 hours Reference the order or catalog number on your invoice and check the corresponding part lists Horizontal Electrophoresis System 1 1 Section 1 Introduction Mode
26. xa uonoajold jueJem ay paap si Juswd nba no aun wes ay jejeuwixoldde je Pays out OB WA juUeJem ay os aun Buiddiys smojje s y AE JNO wouy paddiys si juaudinba JnoA app y Wo syaaM OM SUEIS poled AUSUEM ay VSN ALNVIYVM SLINAOYd IMO IIJILLIIOS YIHSIA ONYIHL 9 1 Horizontal Electrophoresis System Thermo Scientific Section 9 Warranty Information E ENCODER 1006 OSI ch 6 0 Nad UONEWJOJU A UBJEM 104 Jo nquysip e20 INOA D IUO WSN Ay SPISINO suonesidde jeloeds pue sones soueuajuleu uonelado AjuelJem Juswd nba uo suonsanb noA Jamsug 0 Apeal 219M 29 4 8 2 04 L Jo epeueo JO YSN 1984 204 008 1 Je Juauyedag Sage eoluysa AnoA jeo aeseajd p nb si 011 Jas Juswd nbs y adukud ulew anuersld pue uonesado uone e sul juswdinba 1ejap Aj njs1e9 sjenuew UOONJ SU paud SOA juswdinba no aojeq uonewJoju von eJedald aus SAISUBYSIdLUOD yym djey 0 Apes SI S91O Sajes OWE B20 INOA s onpoJd Jo sso JO s yOjd 350 0 sobewep uongjiwi joyym Hulpnjoul sabewep jenuanbasuos Jo Pal pu Aue Joj a qei aq jou eys oway AlddV TIVHS ASOdYNd AYINIILYYVd V YOA SSANLIA YO ALITIAVLNYHOYAN JO SAILNVYYVM ON Gal Id YO WHO NAILLIYM YIHLIHM SSILNVYYVM YAHLO TIV 40 NAIN NI ANY SAISNIOXS SI ALNVYYVM SIHL LoNeunNssp goy paddiys ae syed juewsoe dal pue pied abe sod oway o Daum aq snw syed Bulwjojuoo uou je uondo sowjJay jw juswdinba Jo jusuodwoo Aue jo Wnjal JO
27. xchange Port Option D4 BP The buffer exchange port option is used to recirculate the buffer during extended gel runs Recirculation is used to prevent buffer depletion of certain low ionic running buffers for extended runs multiple sample sets or for RNA gels If your unit has the buffer exchange gt port option it will be fitted with two white P buffer port terminals Figure 8 1 and will contain two separate port inserts packaged in a small plastic bag located inside the unit upon Figure 8 1 Port Insert arrival H How these work The inserts are pushed into the attached ports on the side wall of the unit with the black O ring side facing in The insert will snap into place in the port in the open position and is ready to circulate buffer Appropriate tubing is then connected to the small outer ringed ends of the ports for circulation using a separate recirculator or peristaltic pump To close the port which also releases the insert simply press the flat metal button and the insert detaches The port is now in the closed position Note Buffer may also be passed through a heat exchanger A Thermo Scientific Horizontal Electrophoresis System 8 1 Section 8 Optional Equipment Table 8 1 Comb Options for Models D2 and D3 14 Comb Type Catalog No D Standard D D D D D Standard Standard Standard Micro Well Micro Well Standard Standard Micro Well Micro Well D
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