OPERATING MANUAL Horizon® 11.14 #11068020 Horizon® 20.25
Contents
1. 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Once the unit has been determined to be radiation free lt 100dpm cm2 remove all the hazardous and radioactive labels from the unit If the labels cannot be removed deface them Failure to do so may result in a significant delay or refusal of repair If your unit has non removable contamination detectable with a GM meter and not with paper swipes or detectable with paper swipes but after continued washing the dpm cm remains constant and above 100 of a short half life isotope such as 32P it may be stored for ten half lives of isotopic decay and the decontamination procedure repeated Note Units contaminated with non removable long half life isotopes may not be returned If questions still persist please contact Apogee Designs Ltd Attn Electrophoresis Support 101 Kane Street Baltimore MD 21224 USA Phone 443 744 0368 9 to SPM EST Monday through Friday Fax 410 633 3666 Email info apogeephoresis com 6 4 2 NOTICE REGARDING THE RETURN OF APPARATUS PRODUCTS US Federal Regulations In order to comply with US federal regulations and to protect the health and safety of employees it is imperative that all customers read this notice and adhere to the requirements regarding the return of apparatus products The US Department of Transportation the Department of Health and Human Services and the Nuclear Regulatory Commission have strict regulations on the shipment of h2. Return Shipment 6 4 1 Notice Regarding the Return of Apparatus Products 6 4 2 Warranty 7 0 Warranty 7 1 Declaration of Conformity and CE Mark 7 2 Decontamination Declaration 8 0 FIGURES 1 Horizon 11 14 20 25 Apparatus Components 2 Horizon 11 14 20 25 Gel Casting Configuration TABLES 1 10X TAE Electrophoresis Buffer 10X TBE Electrophoresis Buffer Agarose Volume Requirement for Different Gel Thicknesses 10X Sample Loading Buffer Sample Volumes for Horizon 11 14 Apparatus Combs as a Function of Gel Thickness Sample Volumes for Horizon 20 25 Apparatus Combs as a Function of Gel Thickness Nominal Electrophoresis Times for 1 Agarose Gels at Various Voltages a OS eS HORIZON is a registered trademark of Apogee Designs Ltd DELRIN is a registered trademark of E I duPont de Nemours amp Co Tygon is a registered trademark of Norton Company Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 1 0 BEFORE YOU BEGIN 1 1 IMPORTANT INFORMATION Horizon H11 14 and H20 25 electrophoresis units are authorized for laboratory research use only They have not been qualified for use in any human or animal diagnostic or therapeutic application Use for other than the intended use may be a violation of applicable law The H11 14 and H20 25 Horizontal Gel Electrophoresis Apparatus are designed for separation of preparative and analytical quantities of nucleic acids They are suitable for agarose g
3. apogeephoresis com 7 0 WARRANTY 7 1 WARRANTY Apogee warrants apparatus of its manufacture against defects in materials and workmanship under normal service for one year from the date of receipt by the purchaser This warranty excludes damages resulting from shipping misuse carelessness or neglect and does not include breakage of the electrodes or crazing from cleaning with solvents that attack ABS or acrylic Apogee s liability under the warranty is limited to the repair of such defects or the replacement of the product at its option and is subject to receipt of reasonable proof by the customer that the defect is embraced within the terms of the warranty All claims made under this warranty must be presented to within three years following the date of delivery of the product to the customer This warranty is in lieu of any other warranties or guarantees expressed or implied arising by law or otherwise Apogee makes no other warranty expressed or implied including warranties of merchantability or fitness for a particular purpose Under no circumstances shall Apogee be liable for damages either consequential compensatory incidental or special sounding in negligence strict liability breach of warranty or any other theory arising out of the use of the product listed herein In the interest of bettering performance Apogee reserves the right to make improvements to the design construction and appearance without notice 7 2 DECLAR
4. may offer higher resistance thus heating up and risking sparks and fire 6 2 GENERAL SPECIFICATIONS Type H11 14 H20 25 Dimensions W x L x H 21 5 x 31 5 10 5 cm 32 0x 42 5 12 0 cm Gel Dimensions 11 x 14 cm 20x 25cm Maximum gel thickness 10 mm 10 mm Voltage Range 250 VDC Max 250 VDC Max Current Range 4 360 mA 0 5 Max 4 360 mA 0 5 Max Electrode material Pt Nb strip Pt Nb strip Operating Temperature Range 4 30 C 4 30 C Construction ABS acrylic aluminum ABS acrylic aluminum Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 6 3 TECHNICAL SUPPORT AND SERVICE Should you have any problems with this unit please contact Apogee Designs Ltd Attn Electrophoresis Support 101 Kane Street Baltimore MD 21224 USA Phone 443 744 0368 9 to 5PM EST Monday through Friday Fax 410 633 3666 Email info apogeephoresis com 6 4 INSTRUCTIONS FOR RETURN SHIPMENT IMPORTANT Before sending the unit back to us it is absolutely necessary to call our Technical Support department to get authorization to return products e Return only defective devices For technical problems which are not definitively recognizable as device faults please contact Apogee Technical Support e Use the original box or a similarly sturdy one e Label the outside of the box with CAUTION SENSITIVE INSTRUMENT e Please enclose a detailed description of the fault and when or how the problem occur
5. 007014 41007022 41007030 11951043 11951019 11951050 21069067 31006026 21069059 11099025 21069042 Includes all necessary components 6 0 CARE AND HANDLING 6 1 MATERIALS AND CARE Each H11 14 and H20 25 apparatus is fabricated from high quality ABS and acrylic plastic Acrylic and ABS both have very good heat impact and chemical resistance but will not withstand autoclaving Caution Both electrodes are made from Pt Nb strip for durability Use care when cleaning this apparatus to prevent breakage of the electrodes because they are not warranted against breakage All components may be washed with water and a detergent To remove grease and oils use a hexane kerosene or aliphatic naphtha Never use abrasive cleaners window sprays or any fluid that may contain toluene methylene chloride phenol acetone benzene halogenated hydrocarbon solvents or undiluted laboratory alcohols Routine inspection and maintenance will ensure both the safety and the performance of your horizontal gel apparatus For replacement parts call your distributor or Apogee Technical Support e Because of the relatively high voltages that may be used inspect electrical connections and power cords often If power cords show any signs of wear or damage e g cracks nicks abrasions melted insulation or bare wire replace immediately e Examine the electrode banana plugs and connection nuts to ensure that they are free of corrosion or they
6. 1 Kane Street Baltimore MD 21224 USA apogeephoresis com The sample volumes that can be loaded per well for each standard Horizon Apparatus comb are listed in tables 5 and 6 For analytical purposes keep sample volumes to a minimum Generally 1 mm thick combs provide sharper band definition than 2 mm thick combs Table 5 Sample Volumes for Horizon 11 14 Apparatus Combs as a Function of Gel Thickness Comb Thickness Gel Thickness Comb Type Tooth Width mm mm Capacity Well ul 10 Tooth 7 9 v E D He eo AIWA AIV n A ISU Hl i S oo U1 i S 0o JU1 i S 0o 01 sh co 20 Tooth 3 8 Multichannel Pipet Combs 12 Tooth 7 2 24 Tooth 2 7 Note Volumes given are approximate Low percentage gels lt 0 6 and low melting point agarose gels may have lower sample well volumes Ui Ui 3 3 o0o U01 AJU Tooth width and capacity values are for the central preparative well Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Table 6 Sample Volumes for Horizon 20 25 Apparatus Combs as a Function of Gel Thickness Comb Thickness Gel Thickness Comb Type Tooth Width mm mm mm Capacity Well ul 3 1 100 Prep 165 1 600 2 100 12 Tooth 12 7 15 Tooth 9 5 AJ AIU A AIU An A ISU An AIVA A INVA I S oo U1 i S oo U1 I3 oo Aan i S win S 00 oJ BRIN NIN B R 0 30 Tooth 4 7 Multichannel Pipette Combs 21 Tooth 7 2 42 Tooth 2 7 Apogee Electrop
7. 4 I a Designs Lea OPERATING MANUAL Horizon9 11 14 11068020 Horizon9 20 25 421069026 Horizontal Electrophoresis Apparatus H11 14 Shown Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Before You Begin Important Information Safety Warnings Components Operating Instructions Gel Casting Assembly for Gel Casting Gel Casting Procedure Electrophoresis Post Electrophoresis Troubleshooting Guide Applications Considerations for Agarose Gel Electrophoresis Selecting Gel Concentration Preparing Agarose for Gels Preparing Samples and Loading the Gel Using Multiple Combs Considerations for Electrophoresis Buffers Resolution Effects Heat Effects Ethidium Bromide Staining of Double Stranded DNA Gel Photography Related Products H11 14 Accessories and Replacement Parts H20 25 Accessories and Replacement Parts Care and Handling Materials and Care General Specifications Technical Support and Service Instructions for Return Shipment HORIZONTAL APPARATUS OPERATING MANUAL TABLE OF CONTENTS 1 0 1 1 1 2 1 3 2 0 2 1 2 1 1 2 1 2 2 2 2 2 1 3 0 4 0 4 1 4 1 1 4 1 2 4 1 3 4 1 4 4 2 4 2 1 4 2 2 4 3 4 4 5 0 5 1 5 2 6 0 6 1 6 2 6 3 6 4 Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Cleaning and Decontamination for
8. 4 USA apogeephoresis com 10 11 12 13 To use the buffer circulation system especially important when using TAE electrophoresis buffer see Chapter 4 use the supplied tubing barb adapters to connect 6 mm 0 25 in I D tubing to the fittings on the rear of the H11 14 or H20 25 Connect a circulation pump to the tubing and set for low speed Gently remove the comb s To avoid tearing the bottom of the wells gently wiggle each comb to free the teeth from the gel Slightly lift up one side of the comb then the other Rinse each comb with deionized water and wipe dry before storing Remove any trapped air bubbles to ensure that the wells fill completely with buffer Use a micro pipet or automatic pipet to load the samples on the floor of the wells Samples should contain sufficient glycerol or sucrose to be denser than the electrophoresis buffer For sample loading buffer formulation and the loading capacities for each comb relative to various gel thicknesses see Tables 5 and 6 in Chapter 4 Close the safety interlock lid Connect the power cords to the electrophoresis tank and a 250 VDC power supply Connect the positive red lead at the right side of the apparatus and the negative black lead at the left If you are using the buffer circulation feature start the pump Turn on the power supply and select the desired voltage Small bubbles will rise from the electrodes when the unit is properly connected Nominal electr
9. A TBE buffer formulas in tables 1 and 2 is preferable for your specific application To determine the volume of agarose solution required to produce gels of various thicknesses see table 3 Table 1 10X TAE Electrophoresis Buffer Component Amount Concentration Tris base 48 4g 400 mM Na EDTA 2H O 74g 20 mM Glacial acetic acid 17 0 ml Deionized water to1L Note This is a 10X concentration solution Dilute with deionized water prior to use Final pH should be 7 8 at 25 C Table 2 10X TBE Electrophoresis Buffer Component Concentration Tris base 1M Boric acid anhydrous 0 9M Na2EDTAe2H20 Deionized water Note This is a 10X concentration solution Dilute with deionized water prior to use Final pH should be 8 3 at 25 C Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Table 3 Agarose Volume Requirement for Different Gel Thicknesses Gel Dimensions cm Gel Thickness mm Agarose Volume ml Note Volumes given are approximate 1 Add 1g of agarose per 100 ml of 1X TAE or 1X TBE electrophoresis buffer see tables 1 and 2 for buffer formulas in a bottle or Erlenmeyer flask of at least twice the final volume of solution 2 Loosely cap and weigh the flask 3 Dissolve the agarose in electrophoresis buffer by heating in a microwave oven or boiling water bath with occasional mixing until no granules of agarose are visible 4 Weigh the flask and adjust to the original
10. ATION OF CONFORMITY AND CE MARK Note The information outlined in this section applies only to customers located in the European Union EU This laboratory apparatus is identified with the CE mark This mark indicates that the product complies with the following EU Directives and Standards APPLICATION OF COUNCIL DIRECTIVE S 89 336 EEC Electromagnetic Compatibility 73 23 EEC Low Voltage Directive STANDARDS EN 50081 1 1992 Emissions EN 50082 1 1992 Immunity EN 61010 1 1993 Product Safety Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 8 0 DECONTAMINATION DECLARATION RGA Number IMPORTANT Customer Name Institute Address TEL FAX E mail Unit type Serial number DESCRIPTION OF PROCEDURES USED TO DECONTAMINATE UNIT LOOK AT6 4 1 O 1 Gently washed with water and a non abrasive detergent and rinsed with deionized water 2 Using a solution of 5 household bleach in water or 70 ethanol in water the unit was wiped down using a clean cloth or sponge and neutralized with deionized water O 3 To meet various regulatory and safety standards please follow the decontamination procedures given in 6 4 1 if radioactive materials were used with this product This piece of equipment has not been decontaminated Reason O To the best of my knowledge unit is free of chemical biological or radioactive contamination understand that if the equipment is f
11. IES 10 well precision machined white Delrin comb 14 well precision machined white Delrin comb 20 well precision machined white Delrin comb Horizon 11 14 Gel Casting Apparatus Base UVT tray 2 casting dams H11 14 UVT Tray H11 14 Aluminum Casting Dams Power Cord Replacements 1 black amp 1 red 122 cm long H11 14 Pt Nb Electrode Replacement Includes all necessary components 5 2 H20 25 ACCESSORIES 12 well precision machined white Delrin comb 15 well precision machined white Delrin comb 20 well precision machined white Delrin comb 30 well precision machined white Delrin comb H20 25 Gel Casting Apparatus Base UVT tray 2 casting dams H20 25 UVT Tray H20 25 Aluminum Casting Dams Power Cord Replacements 1 black amp 1 red 122 cm long H20 25 Pt Nb Electrode Replacement Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com DESCRIPTION 1 0 mm thick 2 0 mm thick 1 0 mm thick 2 0 mm thick 1 0 mm thick 2 0 mm thick System Each Package of 2 Package of 2 Kit DESCRIPTION 1 0 mm thick 2 0 mm thick 1 0 mm thick 2 0 mm thick 1 0 mm thick 2 0 mm thick 3 0 mm thick 1 0 mm thick 2 0 mm thick 3 0 mm thick System Each Package of 2 Package of 2 Kit CATALOG 11956026 11956059 11956042 11956075 21076013 21076021 11068046 11084019 11068053 11099025 11958061 CATALOG 11953064 11953080 11953072 11953098 41
12. ate the buffer The bottom of the wells were torn when the comb was removed See 2 2 5 for recommended comb removal procedure This is due to the combination of pH drift and high temperature Circulate or remix buffer periodically Reduce the electrophoretic voltage Gel was cast or electrophoresed out of level Use the bull s eye level to verify that the apparatus is level prior to gel casting and electrophoresis Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com PROBLEM COMMENTS S shaped lanes anomalous migration front results in lanes that are not all running at a uniform speed Flaming bands excessive fluorescence appearing as a trail above the band Wiggly or slanting bands bands are not straight lines or parallel to the top edges of the gel Mix the buffer periodically during electrophoresis Switch to a low conductivity high buffering capacity buffer 0 5X TBE Reduce the salt concentration of the sample Connect a pump to circulate the buffer Reduce the amount of DNA in the sample Reduce the amount of protein and or glycerol in the sample Verify that the wells are free of particles and bubbles before and after loading samples Verify that the agarose is completely dissolved before casting gels Remove any particulate matter from the agarose before casting gels Be sure that bubbles are not trapped against the comb during gel
13. azardous materials 49 CFR Part 173 including etiologic agents 49 CFR Part 173 and 42 CFR Part 72 and radioactive materials CFR 49 Part 173 and 10 CFR Part 20 German Law To comply with German law i e 71 StriSchV 17 GefStoffV and 819 ChemG and to avoid exposure to hazardous materials during handling or repair completion of this form is required before equipment leaves your laboratory When equipment is returned for repair evaluation credit or exchange the customer becomes the shipper and must ensure that the item is free of contamination whether chemical biological or radioactive Procedures for decontamination are described above Materials received that have not been properly decontaminated or units which do not have hazard labels such as caution radioactive materials may be decontaminated at the customer s expense approximately 350 and may result in delay or refusal of repair In addition in the case of radioactive contamination Apogee may be required to notify a licensing authority who in turn may be required to notify the customer s licensing authority Please carefully follow the instructions on decontamination and fill out the Decontamination Declaration that follows Place the Decontamination Declaration inside the top flap of the box where it can be immediately noticed by the receiver Any change to this procedure may result in service delay Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA
14. casting All bands appear as doublets each band is represented twice within the same lane Concentrate the sample and use a thin 2 to 3 mm gel with a thin 1 mm comb Prevent gel movement during photography Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 4 0 APPLICATIONS 4 1 CONSIDERATIONS FOR AGAROSE GEL ELECTROPHORESIS 4 1 1 SELECTING GEL CONCENTRATION The choice of agarose concentration for a gel depends on the range of fragment sizes to be separated The typical agarose concentration is 0 396 to 2 096 Large DNA fragments require low percentage gels while small DNA fragments resolve best on high percentage gels Gels containing 0 596 agarose are very weak and should be electrophoresed at a low temperature 4 C For routine electrophoresis 0 7596 to 1 096 agarose gels provide a wide range of separation 0 15 to 15 kb For a more complete treatment of factors that affect the separation of nucleic acids in agarose gels see Section 4 2 Thin 2 to 3 mm thick and low percentage agarose gels yield better photographs than thick or high percentage gels which exhibit increased opaqueness and auto fluorescence 4 1 2 PREPARING AGAROSE FOR GELS The following protocol yields a 196 w v agarose gel Varying the amount of agarose added in step 1 will produce gels of higher or lower concentration See Section 4 2 to determine whether Tris acetate EDTA TAE buffer or Tris borate EDT
15. ctrophoresis times for agarose gels in TBE and TAE buffers at various voltages are listed in table 7 Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Note Electrophoretic procedures originally developed with Models H4 and H5 often generate additional heat when performed with Horizon Apparatus At higher voltages these heat effects can melt the agarose gel Additional buffer circulation and cooling steps may be necessary to adapt such procedures for use with Horizon Apparatus To prevent drying of the gel and ensure an even voltage gradient across the gel bed submerge the gel with electrophoresis buffer to a depth of only 1 to 2 mm Submerging the gel at a depth gt 2 mm is unnecessary and increases electrical current and heat Table 7 Nominal Electrophoresis Times for 196 Agarose Gels at Various Voltages Electrophoresis voltage Time in Hours for the Time in Hours for the V Buffer 11 14 20 25 TBE TAE 0 5X TBE TAE TBE TAE 0 5X TBE TBE TBE TAE 0 5X TBE Note Values were determined with the gel submerged 1 5 mm the operating current ranged from 4 to 360 mA Current and electrophoresis time vary with buffer volume gel thickness and applied voltage e Formulations for TAE and TBE electrophoresis buffers are listed in tables 1 and 2 e Values represent the time required for BPB dye to migrate 13 cm from the origin in the H11 14 and 22 cm in the H20 25 In a 1 agarose gel BPB comig
16. dams be sure dams are seated properly see Section 3 1 Allow the agarose to cool until thoroughly solidified usually 15 to 30 min To store gels prior to electrophoresis gently remove gel casting dams and comb s wet the gel surface with a small amount of electrophoresis buffer and wrap the UVT tray with the gel still in Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com place with plastic wrap or seal in a plastic bag Store at 4 C Gels can be stored for 1 to 2 days or longer if well sealed Well Forming Comb UVT Gel Tray Casting Dams Figure 2 H11 14 and 20 25 Gel Casting Configuration 2 2 ELECTROPHORESIS 1 Remove the gel casting dams rinse them with deionized water and wipe them dry before storing 2 When transferring a gel from the Horizon 11 14 or 20 25 Gel Casting System verify that the UVT tray is oriented so that the sample wells are in the desired alignment Check that the sample wells line up over one of the well visualization strips and that the UVT tray is seated flush on the tray support platform Note Nucleic acids will migrate toward the positive red electrodes at the right side of the apparatus 3 Pour sufficient electrophoresis buffer into the electrophoresis tank to cover the gel to a depth of 1to2 mm This requires 700 ml for the H11 14 Apparatus and 1 55 L for the H20 25 Apparatus Apogee Electrophoresis 101 Kane Street Baltimore MD 2122
17. e plugs to seal the buffer circulation ports if recirculation is not used One bull s eye level One pair of 122 cm Red amp Black power cords One instruction manual Many of these components are also available separately This chapter provides operating instructions for the H11 14 and H20 25 Apparatus Refer to Chapter 4 for information on commonly used buffers agarose concentrations sample volumes and post electrophoresis handling of the gel Review Figure 1 to identify the features and components discussed in these instructions Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Casting Dams Comb Comb Alignment Slots Casting Dam Slots Pour Spout LevelingFeet Figure 1 H11 14 and 20 25 Components Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 2 0 OPERATING INSTRUCTIONS 2 1 GEL CASTING Note If you are using gels cast in advance with the Horizon 11 14 or 20 25 Gel Casting System proceed to Section 2 2 2 1 1 ASSEMBLY FOR GEL CASTING 1 Place the apparatus on a flat level surface Open the safety interlock lid Place the UVT tray in the electrophoresis tank on the tray support platform The red well visualization strips should be visible through the tray Slide the gel casting dams simultaneously into the V grooves in the electrophoresis tank Make sure that the tops of the dams are level Apply even gentle downward pres
18. el electrophoresis procedures Because of the double wall construction of the H11 14 and H20 25 Apparatus the heat generated by electrophoresis does not dissipate as quickly as with Models H4 and H5 With the Horizon Apparatus gels electrophoresed at the same voltage will be warmer and samples will move faster These effects are particularly evident at higher voltages and may necessitate buffer cooling steps to avoid melting the gel Consult Chapter 4 Table 7 for further information If the product is used in a manner not specified by Apogee the protection provided by safety features of the product may be impaired Please carefully follow the manual s instructions Do not alter equipment or operate with broken components Failure to adhere to these directions could result in personal and or laboratory hazards as well as invalidate the equipment warranty 1 2 SAFETY WARNINGS e CAUTION SHOCK HAZARD Although equipped with a safety interlock system this apparatus should always be operated with extreme caution Careless handling could result in electrical shock The power supply should have open circuit sensing e This apparatus should always be operated with caution Careless handling can result in electrical shock e The system should be operated by trained personnel only e Some reagents indicated for use in this manual may be hazardous e g ethidium bromide acetic acid and boric acid etc exercise care with these reagents e A
19. g common problems are given below Should these suggestions not resolve the problem please call Technical Support see Section 6 3 for numbers If the unit must be returned for repair also contact our service department the technical support or your local distributor for shipping instructions Please include a full description of the problem PROBLEM COMMENTS Bubbles do not appear on the electrodes when DC voltage is connected Electrodes turn gray Agarose solution leaks during casting the gel casting dams are clean BPB dye turns yellow pH change during electrophoresis Results are uninterpretable Samples leak underneath the gel upon loading Gel melts or becomes soft near sample wells Pronounced smiling along one edge of the gel occurs corresponding bands in different lanes migrate slower toward one edge Verify that the DC power supply is operating properly Verify continuity of the power cords with an ohmmeter This occurs under normal operating conditions Performance is not affected Verify that the sealing surfaces of the UVT tray and Verify that the gel casting dams are properly seated Verify that the ends of the UVT tray are flat and free of nicks Cool the agarose to 50 C to 60 C before pouring Check the pH of the electrophoresis buffer refer to tables 1 and 2 Be sure to use Tris Base and not Tris HCl Mix the buffer periodically during electrophoresis Connect a pump to circul
20. h water and a non abrasive detergent and rinsed with deionized water Dry using a soft cloth paper towel or allow to air dry A light application of hexane kerosene or aliphatic naphtha will remove grease To prevent surface damage never use abrasive cleaners window sprays or scouring pads to clean these products Avoid excessive exposure to UV light phenol acetone benzene halogenated hydrocarbon solvents or undiluted alcohols because they may cause crazing STEP 2 BIOLOGICAL CLEANING PROCEDURE Using a solution of either 5 household bleach in water or 70 ethanol in water wipe down the apparatus using a clean cloth or sponge Neutralize the solution by wiping the surface with a mild nonabrasive detergent and rinse well with water STEP 3 RADIOLOGICAL DECONTAMINATION PROCEDURE To meet various regulatory and safety standards please follow the decontamination procedure given here if radioactive materials are used with this product or are used in the vicinity of where this apparatus has been used or stored WARNING We cannot and will not accept return of products that are contaminated with any radioactivity For beta emitting isotopes such as 32P use a GM type radioactivity meter calibrated in counts per minute CPM to determine the background readings for your work area Wearing latex gloves survey the unit to be returned with the GM meter If any part of the unit is found to show readings higher than background
21. horesis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com veJ n By 02 Note Volumes given are approximate Low percentage gels lt 0 6 and low melting point agarose gels may have lower sample well volumes Tooth width and capacity values are for the central preparative well 4 1 4 USING MULTIPLE COMBS The multiple comb alignment slots in the Horizon 11 14 and 20 25 Apparatus lend themselves to a variety of applications For example using two rows of wells on the same gel doubles the number of samples of mini prep plasmid DNA that can be screened A row of wells at the bottom of the gel provides a convenient way to include quantitative standards on a gel for Southern blot hybridization Note To use this feature add the standards to the bottom row and let them migrate into the gel for just a few minutes before electrophoresis is complete 4 2 CONSIDERATIONS FOR ELECTROPHORESIS BUFFERS 4 2 1 RESOLUTION EFFECTS For electrophoresis of agarose gels of the same concentration and at a fixed voltage TAE buffer provides better resolution of fragments gt 4 kb in length while TBE buffer offers better resolution of 0 1 to 3 kb fragments TBE has a higher buffering capacity and lower conductivity than TAE and is therefore better suited for high voltage gt 150 V electrophoresis TBE buffer also generates less heat at an equivalent voltage and does not allow a significant pH drift Note Because of its lower buffe
22. lways follow the power supply manufacturer s recommendations for use and follow safety procedures e Always turn off the DC power source before disconnecting the power cords from the apparatus e Never operate damaged or leaking equipment Inspect the apparatus electrical connections and power cords prior to use e For maximum safety always operate this apparatus in an area that is not accessible to unauthorized personnel Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 1 3 COMPONENTS The H11 14 and H20 25 Apparatus are designed for simplified gel casting and electrophoresis These apparatus are engineered for durable performance and easy storage Refer to Figures 1 and 2 to identify the following features and components One ABS electrophoresis tank with clear acrylic safety interlock lid adjustable leveling feet self sealing ports for buffer circulation molded V grooves for placement of gel casting dams and a tray support platform with red well visualization strips and black centimeter graduations One UVT tray 11 x 14 cm or 20 x 25 cm gel bed with multiple positioning slots for well forming combs One pair of aluminum gel casting dams One precision machined Delrin well forming comb o with H11 14 Apparatus one 14 tooth 1 mm thick o with H20 25 Apparatus one 20 tooth 1 mm thick One pair of tubing barb adapters for use with 6 mm 0 25 in I D recirculation tubing One pair of pip
23. ophoresis times for TAE and TBE buffers are listed in Table 7 in Chapter 4 Monitor electrophoresis by following the migration of the bromophenol blue BPB dye Movement should be in the direction of the positive electrodes at the right side of the apparatus Use the black 1 cm graduations visible below the UVT tray to determine approximate migration rates When electrophoresis is complete turn off the power supply Disconnect the power cords from the power supply and the apparatus Turn off the circulation pump 2 2 1 POST ELECTROPHORESIS 1 2 Open the safety interlock lid Lift out the UVT tray and gel Slide the gel out of the UVT tray for staining or subsequent analysis for further information Remove the gel with care agarose gels tear easily if not properly supported Properly discard the electrophoresis buffer Use the pouring spouts at the front corners of the unit to transfer the buffer to a waste receptacle Do not reuse the buffer Disconnect the buffer circulation pump by pressing the metal tab on each buffer port to release the quick connect fittings Thoroughly rinse the electrophoresis tank quick connect fittings and tubing with deionized water Remove any residual agarose from the UVT tray by rinsing with deionized water Wipe dry or allow to air dry before storing Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 3 0 TROUBLESHOOTING GUIDE Some suggestions for resolvin
24. ound to be contaminated regardless of the signature on this document the equipment may be decontaminated at my expense Also if the equipment is found to be contaminated the response time for repairs will be delayed Signature Title Date Please place completed and signed form inside the box with the equipment where it can immediately be noticed by the receiver We appreciate you taking the time to perform the necessary precautions to ensure that equipment being returned can be safely handled by our employees Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com
25. rates with DNA fragments of approximately 200 bp in 1X TBE buffer and 400 bp in 1X TAE buffer 4 3 ETHIDIUM BROMIDE STAINING OF DOUBLE STRANDED DNA To visualize double stranded DNA after electrophoresis the gel should be transferred from the UVT tray to a 0 5 ug ml solution of ethidium bromide in deionized water Approximate staining time is 10 to 15 min for a 3 mm thick gel and longer for thicker gels As an optional subsequent step to reduce background fluorescence the gel can be destained in deionized water for 15 to 30 min Alternatively ethidium bromide may be added directly to the agarose prior to casting so that the gel is electrophoresed in the presence of ethidium bromide However this procedure reduces the migration rate and may alter the relative electrophoretic mobility of nucleic acids reference 3 Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 4 4 GEL PHOTOGRAPHY A darkroom or light tight enclosure camera digital camera and UV light source are required for photography of gels stained with ethidium bromide For best results place the stained gel directly on top of a 300 nm or 254 nm transilluminator If the camera contains ASA 3000 or equivalent film the required exposure at maximum aperture f 4 5 should be between 1 4 and 2 seconds The intensity of the light source distance between the gel and the camera lens film or shutter speed lens aperture and choice of photographic filte
26. red Important Clean all parts of the instrument from residues and of biologically dangerous chemical and radioactive contaminants Please include a written confirmation use the respective Decontamination Declaration Certificate following in Section 8 that the device is free of biologically dangerous and radioactive contaminants in each shipment If the device is contaminated it is possible that Apogee will be forced to refuse to accept the device The sender of the repair order will be held liable for possible damages resulting from insufficient decontamination of the device Please enclose a note which contains the following 1 Sender s name and address and 2 Name of a contact person for further inquiries with telephone number 6 4 1 CLEANING AND DECONTAMINATION FOR RETURN OF PRODUCTS Use the original product packaging whenever possible to avoid damage to the unit being returned All returned material must be cleaned and decontaminated prior to shipping The components of apparatus products are fabricated from a variety of materials including ABS acrylic vinyl glass silicone aluminum and stainless steel Please clean any unit or product to be returned using the following three step procedure Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com STEP 1 GENERAL CLEANING PROCEDURE For materials not contaminated with biological or radiological substances components may be gently washed wit
27. ring capacity TAE requires circulation or mixing periodically for full length electrophoresis particularly at higher voltages Band compression of fragments of high molecular weight gt 5 kb occurs as voltage increases This effect is observed with both TBE and TAE buffers Band definition remains sharp even above 200 V provided that the gel is not over loaded Linear DNA fragments from 0 15 to 10 kb 25 ng total are easily resolved ona 0 8 agarose gel in 0 5X TBE buffer electrophoresed for 30 min at 200 V TAE buffer provides better results for analysis of supercoiled DNA Anomalous migration of supercoiled DNA particularly with high molecular weight gt 7 kb fragments occurs when TBE buffer is used at gt 75 V Use of TBE buffer also reduces the ability to resolve supercoiled DNA from nicked circular and linear DNA in the absence of ethidium bromide For accurate size determination with supercoiled DNA supercoiled DNA of known sizes must be electrophoresed in an adjacent lane of the gel 4 2 2 HEAT EFFECTS Electrophoresis at high voltages generates heat and high conductivity buffers such as TAE generate more heat than low conductivity buffers Caution should be exercised in agarose gel electrophoresis at gt 175 V Heat buildup can cause gel artifacts such as S shaped migration fronts and in prolonged electrophoresis can melt the agarose gel Low melting point agarose gels should never be electrophoresed at high voltages Nominal ele
28. rs will all affect the exposure time Use of a 300 nm transilluminator allows gels to be photographed while in place in the UVT tray although this will increase the required exposure time Transmitted UV light yields the highest sensitivities 1 ng of DNA in a 5 mm wide band in photographing gels Photography under incident UV light is approximately 10 times less sensitive A UV blocking filter Kodak 2B Wratten filter used in conjunction with a red gelatin filter Kodak 23A Wratten filter provides the highest contrast Due to the fluorescence of the 2B filter the two filters must be oriented so that the red 23A filter is adjacent to the camera lens The ethidium bromide DNA complex fluoresces at 590 nm upon excitation at 302 nm 2 Short wave 254 nm sources provide an equivalent level of sensitivity however high energy UV causes photodimerization and nicking of the DNA Long wave transilluminators 366 nm are much less efficient REFERENCES 1 Maniatis T Fritsch E F and Sambrook J 1982 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor New York 2 Rickwood D and Hames B D eds 1982 Gel Electrophoresis of Nucleic Acids A Practical Approach IRL Press Oxford England 3 Longo M C and Hartley J L 1986 LTI Focus 8 3 Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 5 0 RELATED PRODUCTS AND REPLACEMENT PARTS 5 1 H11 14 ACCESSOR
29. sure to both dams to seat the sealing surfaces of the dams against the ends of the UVT tray Do not force the dams down since this can displace the tray out of level Place the bull s eye level in the center of the UVT tray and turn the adjustable feet as needed to level the apparatus Insert a comb or combs into the preferred alignment slots of the UVT tray The teeth of each comb should line up over one of the well visualization strips Ensure that each comb rests unobstructed and squarely in its slots The apparatus is now ready for gel casting figure 3 Note Nucleic acids will migrate toward the positive red electrodes at the right side of the Horizon Apparatus 2 1 2 GEL CASTING PROCEDURE 1 Prepare the desired volume of molten agarose in electrophoresis buffer in a bottle or Erlenmeyer flask For information on agarose preparation buffer formulation and the effects of varying gel concentration and volume see Chapter 5 Loosely cap the container Allow the molten agarose to cool to 50 C to 60 C Caution Casting gels with agarose above 60 C will result in poor sealing at the gel casting dams and may cause the bottom of the UVT tray to bow Pour the measured volume of molten agarose into the center of the UVT tray Use a pipette tip to distribute the agarose evenly over the surface of the UVT tray and to remove any air bubbles particularly from around comb teeth Note If molten agarose leaks from below gel casting
30. wash the area using Radiacwash Atomic Products Corp and paper towels or another similar commercially available detergent If none are available a mild detergent or a Formula 409 like solution will do As you clean discard liquid and solid waste gloves and paper towels according to your local and institutional regulations for radioactive material disposal Continue washing until the GM meter reading for the contaminated area s is equal to or below background To decontaminate units where a GM meter is not as useful for detection as with H or S it will be necessary to perform swipes of the unit and detect using a scintillation counter The unit should be dry Wipe surfaces with dry paper circles these are commercially available or you can make your own Areas can be charted so that individual swipes can be done on different surfaces to better isolate areas of contamination Swipes should be placed into individual scintillation vials with an appropriate floor and then analyzed on a properly programmed scintillation counter If contamination above 100 disintegrations per minute dpm 100cm dpm CPM efficiency is found wash the area as described above in 33P decontamination After cleaning the area swipe it a second time to determine the amount of contamination remaining If the area still has greater than 100 dpm cm2 continue the cycle of swipes and washing until you achieve a reading of less than 100 dpm cm Apogee Electrophoresis
31. weight with deionized water to compensate for evaporation 5 Put the capped flask in a water bath at 50 C to 60 C and allow the agarose to equilibrate at that temperature before pouring gels 4 1 3 PREPARING SAMPLES AND LOADING THE GEL The amount of DNA that can be loaded per well is variable and depends upon the number and size of the DNA fragments and the cross sectional area of the well well width x gel thickness As a general rule the minimum amount of DNA detectable by ethidium bromide staining is 1 ng in a 5b mm wide band on a 3 mm thick gel For preparative purposes on a 3 mm thick gel the amount of DNA loaded should not exceed 50 ng per 5 mm wide band Overloading the gel may cause trailing and distortion of bands Table 4 contains a formula for a sample loading buffer which should be added to DNA samples prior to loading For alternative formulas for sample loading buffers see Section 4 4 References 1 and 2 Table 4 10X Sample Loading Buffer Component Amount Concentration Glycerol 5 ml 5096 v v Na2EDTA 2H20 0 37g 100 mM Sodium dodecyl sulfate 0 1g 196 w v Bromophenol blue 0 01 g 0 196 w v Deionized water to 10 ml Note This is a 10X concentration solution Add 0 1 volume of buffer to samples and apply directly to gel If the samples contain l cohesive ends as with DNA restriction fragments the samples in buffer should be heated at 65 C for 5 to 10 min prior to loading Apogee Electrophoresis 10
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