OPERATING MANUAL Model S2® #21105036
Contents
1. f c Electrophoresis OPERATING MANUAL Model S2 21105036 Sequencing Gel Electrophoresis Apparatus Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com S2 SEQUENCING APPARATUS OPERATING MANUAL TABLE OF CONTENTS Before You Begin Important Information Safety Warnings Components Operating Instructions Gel Casting Preparation of Glass Plates Using Gel Sealing Tape Preparation of Glass Plates Using the S2 Casting Clamp Pouring the Gel Using Gel Sealing Tape Pouring the Gel Using the S2 Casting Clamp Electrophoresis Pre electrophoresis Loading Samples Electrophoresis Post Electrophoresis Troubleshooting Guide References Related Products and Replacement Parts Care and Handling Materials and Care General Specifications Technical Support and Service Instructions for Return Shipment Cleaning and Decontamination for Return Shipment Notice Regarding the Return of Apparatus Products Warranty Warranty Declaration of Conformity and CE Mark Decontamination Declaration Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 1 0 1 1 1 2 1 3 2 0 2 1 2 1 1 2 1 2 2 1 3 2 1 4 2 2 2 2 1 2 2 2 2 2 3 2 2 4 3 0 4 0 5 0 6 0 6 1 6 2 6 3 6 4 6 4 1 6 4 2 7 0 7 1 7 2 8 0 FIGURES NOW PWNE S2 Gel E2. i e 71 StriSchV 17 GefStoffV and 19 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 that 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 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
3. Pasteur pipette to wash away urea that has diffused into the wells Note Not rinsing the wells will prevent samples from layering properly on the gel surface 4 Using a sharkstooth comb to form shallow wells see Section 2 1 3 allows you to load samples with a micropipette or a drawn out capillary pipette Figure 6 When using the sharkstooth comb provided with the Model S2 Apparatus load 2 to 3 ul of Figure 6 Sharkstooth Comb sample per well When using accessory double fine Sample Loading Technique sharkstooth combs load 1 5 to 2 ul of sample 5 When using optional square toothed combs load samples onto the bottom of the wells with capillary pipettes To determine the sample loading volumes of Model S2 Apparatus square toothed combs relative to gel thickness see Table 3 Table 3 Sample Loading Volumes f or Model S2 Apparatus Square Toothed Combs as a Function of Gel Thickness Number of Teeth Tooth Width mm Gel Thickness mm Capacity Well ul 16 20 32 11 5 7 0 4 25 1 6 40 Note All loading volumes are calculated for a comb insertion depth of 5 mm Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 2 2 3 ELECTROPHORESIS After loading the samples close the upper and lower safety lids and connect the DC power cords to the sequencing apparatus and the DC power supply 1 3 Warning All banana plug connections must be fully seated to prevent potential
4. different rates across the gel Verify that the top integral gel clamps are firmly but not overly tightened The ribs of the silicone gasket should be slightly and evenly compressed but not wrinkled First loosen the integral gel clamps a partial turn If the leaking does not stop try tightening the clamps carefully Verify that the black foam blocks are securely seated against the short glass plate before casting the gel Check the silicone gasket for tears or other damage Verify that a pinhole leak or crack has not developed in the glue joints of the upper buffer reservoir Buffer is leaking from the upper buffer chamber See preceding comments CAUSES OF COMMON PROBLEMS IN SEQUENCING GEL ELECTROPHORESIS PROBLEM POSSIBLE CAUSE SUGGESTED SOLUTION Blurry bands Urea diffusing into the wells After pre electrophoresis wash the urea from the wells before loading the samples Formamide in sample buffer degraded Remake sample buffer with fresh formamide Excessive salt in the sample Precipitate DNA with ethanol and ammonium acetate Radiolabel properties lead to scattering Try 35S or 33P labeled dNTP s instead of 32P of signal Gel was overloaded Urea diffused out of gel labeled dNTP Label the sequencing primer with T4 polynucleotide kinase instead of internal labeling Use a shorter exposure Use cassette that assures tight contact of gel to film Do not use an intensifying screen Load l
5. 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 22P 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 hazardous 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
6. reinsertion of the comb in the sample loading teeth down configuration and to allow loading with a micropipette Mark one end of the comb so that you can keep it in the same left to right orientation when loading samples later If you are using a DELRIN square toothed comb available separately insert the teeth 4 to 5 mm below the edge of the short plate 5 Atop clamp and spring clips are recommended for securing the comb plate sandwich Place two spring clips over the edge of the plates to clamp on the comb Figure 4 Do not place the pressure points of the spring clip over the side spacer Place the top clamp over the top of the glass plates and clamp the center of the top edges of the plate securely against the comb Proper placement of the spring clips and top clamp will force the plates against the comb ensuring a tight fit of the comb following polymerization If you use spring clips along the lower edge of the gel for sealing purposes clamp them on the side spacers Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Note If spring clips are not put in place during polymerization the comb will not fit tightly which increases the possibility of leaks between wells Figure 4 Gel Casting Configuration of Sharkstooth Comb 6 Leave the glass plates in their near horizontal position until the acrylamide has polymerized 7 Remove the spring clips and top clamp if used and tape Carefully
7. slide the comb from between the glass plates 8 Rinse the top of the gel with electrophoresis buffer to remove any unpolymerized acrylamide Rinse the comb with deionized water 9 If you are using a sharkstooth comb reinsert the comb between the glass plates with the teeth toward the gel Insert the comb until the teeth just make contact with the surface of the gel figure 5 Do not allow the teeth to pierce the gel A very slight indentation of the gel should be visible when the comb is properly inserted Do not slide the comb laterally after it has come in contact with the gel 2 1 4 POURING THE GEL USING THE S2 CASTING CLAMP The S2 Casting Clamp allows for the gel to be filled from either the top or the bottom of the assembled plate sandwich To cast the gel from the top follow the ADDISPPINININDINIIINIME instructions below making sure that the Pai her M A g Ll AGMA S AESC LS AER UE bottom fill port is sealed LAC VASES SLL SSS LL SL LLL HZ VALLE FDE LL LARV PP Uf EO LA A 1 To fill the gel from the top use a D d A dg ie ko TAMO E large syringe squirt bottle funnel RN or beaker to pour the gel solution N N Y iv between the plate sandwich o Hold the assembled plate WY sandwich at a 25 35 angle on UK Figure 5 Sample Loading one bottom corner so that the gel Configuration of Sharkstooth Comb solution flows evenly down along the lower side spacer Maintain a constant even flow to reduce the ch
8. sparking and fire hazards Turn on the power supply and set the voltage or wattage to the proper setting for the gel see Table 2 for recommended DC power settings for various gel thicknesses Monitor the progress of electrophoresis by following the migration of a marker dye front or by any other preferred method When electrophoresis is complete turn off the power supply and disconnect both DC power cords first from the power supply and then from the sequencing apparatus 2 2 4 POST ELECTROPHORESIS 1 Open the buffer chamber drain valve to allow the buffer in the upper chamber to drain into the covered rear chamber of the lower buffer tray Open the upper safety lid and rinse the upper buffer chamber with 25 to 50 ml of deionized water to prevent buffer crystallization in the internal drain tubing Release the integral gel clamps by loosening the screw knobs and remove the gel sandwich from the sequencing apparatus Lay the gel sandwich flat with the short glass plate on top on a piece of absorbent paper Open the lower safety lid and remove the lower buffer tray from the sequencing apparatus The buffer in the open front chamber of the tray will contain any radioactive nucleotides electrophoresed out of the gel Dispose of this liquid by pouring away from the drain hole in the covered rear chamber of the tray The buffer in the rear chamber drained from the upper chamber may then be discarded separately After disposing of all b
9. ance of forming bubbles in the solution Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com As the gel sandwich fills gradually lower the casting clamp until it rests on the molded feet approximately a 5 angle The gel mold should be slightly overfilled to ensure complete polymerization at the top Before the acrylamide polymerizes insert a well forming comb into the top of the gel If you are using a sharkstooth comb insert the flat edge of the comb between the glass plates to a depth of 2 to 3 mm below the top of the short glass plate Figure 4 Keep the flat edge shallow to simplify subsequent reinsertion of the comb in the sample loading teeth down configuration and to allow loading with a micropipette Mark one end of the comb so that you can keep it in the same left to right orientation when loading samples later If you are using a DELRIN square toothed comb available separately insert the teeth 4 to 5 mm below the edge of the short plate For improved comb sealing clamp the glass plate sandwich over the comb with a top clamp over the center of the comb It is important for the top clamp to be placed over the comb only Clamping unsupported glass will distort the thickness of the gel The casting clamp does not require the use of accessory spring clips along the sides of the gel sandwich Leave the glass plates in their near horizontal position until the acrylamide has polymerized Remove t
10. ase 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 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 type solution will d
11. ays or any fluid that may contain toluene methylene chloride phenol acetone benzene halogenated hydrocarbon solvents or undiluted laboratory alcohols The following simple routine inspection and maintenance procedures will help ensure both the safety and the performance of the Model S2 Apparatus For ordering information for replacement parts refer to Chapter 5 For replacement parts call your distributor or Apogee Technical Support 1 Because ofthe 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 2 If the banana plug connectors to the unit or the power supply do not exhibit reasonable friction or can be rocked easily replace the plugs or power cords 3 Examine the electrode banana plugs and connection nuts to ensure that they are free of corrosion or they may offer higher resistance thus heating up and risking sparks and fire If connection nuts are corroded they should be replaced 4 Regularly inspect the silicone gasket for cuts or tears and replace as needed 5 After each use rinse the aluminum plates thoroughly with deionized water to remove salts and urea Failure to keep plates properly clean may affect performance and introduce a potential electrical hazard 6 2 GENERAL SPECIFICATIONS Type Model S2 Dimensions W x L x H 42 2 x 21 6 x 44 5 c
12. 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 DECLARATION 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 T
13. d grease Hold the casting clamp vertically with your thumbs resting at the top of the molded handles and your index fingers over the molded feet flex the clamp outward in a bow like manner so that the bottom of the clamp can fit onto the bottom of the gel sandwich Fit the gel sandwich snugly into the bottom of the casting clamp Start at one corner and seal towards the other corner After the gel sandwich is seated into the bottom of the clamp fit the sides of the clamp onto the sides of the gel sandwich Start at the bottom of one side using your hands to smooth the clamp onto the gel sandwich Repeat this for the other side of the gel sandwich When properly placed the sides of the casting clamp will extend to within 4 5 mm of the top of the short glass plate See Figure 3 If the sides of the clamp should extend further than this reposition the sides of the casting clamp so that they are at the top of the short glass plate Glass plates CLE 2 i I Casting clamp Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 2 1 3 POURING THE GEL USING GEL SEALING TAPE 1 Prepare the acrylamide gel solution see Table 1 for the approximate volumes required for different gel thicknesses Note Most gel formulations allow approximately 10 min before polymerization Polymerization time can be extended by reducing the amount of TEMED added Warning Acrylamide is a known neurotoxin Cons
14. e are free of nicks or chips Clean the glass plates thoroughly with a nonabrasive detergent and a plastic scouring pad The cleaning solution should not leave a soap residue when rinsed thoroughly Avoid scratching the surface of the glass plates Note Never use steel wool to scrub the glass plates Rinse the glass plates thoroughly with deionized water and wipe dry If desired treat the inside acrylamide contact surface of one or both glass plates usually the short plate with a siliconizing reagent following the manufacturer s instructions Immediately before assembling the glass plate sandwich rinse the inside surfaces with ethanol and wipe them dry with a lint free paper towel Avoid touching the inside surfaces with your fingers Assemble the glass plate sandwich in the conventional manner place the long glass plate inside up on the bench align the vinyl side spacers at the sides and place the short glass plate inside down on the side spacers Make sure that the foam blocks at the tops of the side spacers are snug against the top of the short glass plate and that the ends of the side spacers are 4 to 7 mm short of the bottom of the assembled plate sandwich Note Before using vinyl side spacers for the first time attach a foam block at one end of each spacer Remove the protective backing from the adhesive side of a foam block align the end of the block with the end of the side spacer and press the block firmly into place F
15. e gel sealing tape 38 mm 1 50 wide x 66 m 72 yards long e Instruction manual Many components are also available separately Refer to Chapter 5 Related Products for ordering information Please read all of the following instructions before using the Model S2 Apparatus Review Figure 1 below to identify features and components discussed in these instructions Safety lid Sharkstooth combs Vinyl side spacer with foam block f Silicone gasket eR Upper buffer drain valve I Long glass plate Integral gel clamps Safety lid Gel supports Removable buffer tray selbst d 7 DC HV power cords Figure 1 Model S2 Components Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 2 0 OPERATING INSTRUCTIONS This chapter provides operating instructions for the Model S2 Apparatus For a list of references containing formulations of buffers and acrylamide solutions as well as other applications information for polyacrylamide gel sequencing and fragment purification see Chapter 4 References 2 1 GEL CASTING Gels can be cast after assembling the glass plates and spacers using Gel Sealing Tape or the S2 Casting Clamp These techniques are appropriate for casting polyacrylamide gels 2 1 1 PREPARATION OF GLASS PLATES USING GEL SEALING TAPE 1 Select a pair of glass plates one long one short Be sure that the edges to be sealed with tap
16. e top of the gel Clean the flat edge of the sharkstooth comb Insert sharkstooth comb only to the point of dimpling the surface of the gel Electrophorese with constant voltage or power not constant current Do not exceed settings that yield a gel surface temperature of 45 50 C Use a buffer gradient gel Use wedge spacers Do a double loading Use ammonium persulfate powder to make solution Alternatively remove the ammonium persulfate from the capsule before dissolving 12 Allow the gel to polymerize at a 5 angle instead of flat Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com PROBLEM POSSIBLE CAUSE SUGGESTED SOLUTION Gel temperature is not uniform Frowning gels Improper clamping during casting Corresponding bands in outer lanes electrophorese faster than inner lanes Lanes curve outward toward bottom of the gel Improper gel casting Bands are not Gel sandwich is not level even from lane to lane Use an electrophoresis unit with an aluminum plate to disperse heat evenly Apply the pressure points of the binder clips only over the spacer Polymerize the gel at an angle of lt 10 Do not over tighten clamps on the apparatus Improper clamping of the gel into the electrophoresis chamber Make sure that spacers are pushed against the polymerized acrylamide Do not use bottom clamps without a bottom spacer Do not pull plates t
17. eplacement Includes all necessary components S2 Lower Buffer Pt Ti Electrode Replacement Includes all necessary components Upper Buffer Chamber Banana Plug Replacement Includes cap nut O ring and Banana Plug Lower Buffer Chamber Banana Plug Replacement Includes cap nut O ring and Banana Plug Gel Clamp Replacement Pt Ti electrode wire A Flat washer Wire carrier Acorn nut Figure 7 S2 Repair Components Description Package of 2 Kit Kit Kit Kit see Figure 7 Kit see Figure 7 Kit Banana plug Gel clamp 11032018 11032026 Catalog 11099025 21105358 21113014 11114014 21105127 21105101 11958352 Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 6 0 CARE AND HANDLING 6 1 MATERIALS AND CARE The S2 is the world standard manual sequencing apparatus Every S2 apparatus is machined and fabricated from high quality aluminum ABS acrylic and polycarbonate 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 Ti ire 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 spr
18. ess sample Do not store precast gels under electrophoresis buffer overnight in the sequencing unit Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com PROBLEM POSSIBLE CAUSE SUGGESTED SOLUTION Smeared Sample electrophoresing with the ion bands in front Gel temperature too high Sample electrophoresing at the surface of the acrylamide instead of through gel Formamide in sample buffer degraded Excessive salt in the sample Wavy bands Surface of the gel did not polymerize evenly Sharkstooth combs punctured the surface of the gel Glass plates Gel is too hot crack Lack of Limitations in the resolving capabilities resolution in of acrylamide the upper half of the gel Capsules used to make 10 ammonium persulfate solution Smiling gels Gel thickness is not uniform Corresponding bands in outer lanes electrophorese slower than inner lanes Pre electrophorese gel until the current starts to drop generally gt 1 2 hour Do not electrophorese gel on constant current Use constant power to maintain gel at 45 50 C Use a surface thermometer during electrophoresis Thoroughly rinse residual siliconizing agent off glass plates Ethanol wipe before using Thoroughly wash new glass plates with a strong lab soap before using Remake sample buffer with fresh formamide Precipitate DNA with ethanol and ammonium acetate Recast gel with extra acrylamide at th
19. he top clamp if used and carefully slide the comb from between the glass plates Remove the glass plates from the casting clamp Pull the casting clamp off the glass plates starting at the top and working your way down Take care in that there may be some unpolymerized acrylamide in the bottom of the casting clamp Rinse the top of the gel with electrophoresis buffer to remove any unpolymerized acrylamide Rinse the comb with deionized water If you are using a sharkstooth comb reinsert the comb between the glass plates with the teeth toward the gel Insert the comb until the teeth just make contact with the surface of the gel Figure 5 Do not allow the teeth to pierce the gel A very slight indentation of the gel should be visible when the comb is properly inserted Do not slide the comb laterally after it has come in contact with the gel To fill the gel from the bottom assemble the gel plates so that when the casting clamp is laying down on the molded feet the short glass plate is on top 1 Place the assembled gel sandwich on a level surface resting on the molded feet of the casting clamp Open the bottom fill port and insert a Luer fitting Attach a syringe of suitable reservoir to the fitting and slowly fill the gel Do not interrupt the filling as this may introduce bubbles into the gel solution Once the gel is filled insert the comb as before and allow the gel to polymerize Apogee Electrophoresis 101 Kane Street Ba
20. his 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 4 E mail Unit type Serial number DESCRIPTION OF PROCEDURES USED TO DECONTAMINATE UNIT LOOK AT6 4 1 O 1 S2 was 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 the decontamination procedures given in 6 4 1 if radioactive materials were used to decontaminate 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 found 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 repa
21. irs 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
22. lectrophoresis Apparatus Components Taping Glass Plates Assembly Using S2 Casting Clamp Gel Casting Configuration of Sharkstooth Comb Sample Loading Configuration of a Sharkstooth Comb Sharkstooth Comb Sample Loading Technique S2 Repair Components TABLES Approximate Gel Solution Volume Requirement for Different Gel Thicknesses Recommended Electrophoresis Power Settings for Different Gel Thicknesses Sample Loading Volumes for Model S2 Apparatus Square Toothed Combs as a Function of Gel Thickness MODEL S2 is a registered trademark of Apogee Designs Ltd DELRIN and MYLAR are registered trademarks 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 The Model S2 Sequencing Gel Electrophoresis Apparatus is authorized for laboratory research use only It has 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 Model S2 is designed for vertical polyacrylamide gel separations of the products of dideoxy and chemical sequencing reactions The system is useful for preparative purifications of oligonucleotides DNA foot printing procedures ribonuclease protection assays and sequence determinations of up to 250 to 350 bases This manual provides operati
23. ltimore MD 21224 USA apogeephoresis com 2 2 ELECTROPHORESIS 2 2 1 PRE ELECTROPHORESIS 1 Before beginning electrophoresis verify that the sequencing apparatus is in a secure and level position 2 Place the gel sandwich in the sequencing apparatus with the short glass plate inward so that the foam blocks on the side spacers form a seal with the blue silicone gasket Rest the bottom edge of the sandwich on the ribbed gel support blocks in the lower buffer tray 3 Secure the gel sandwich with the integral gel clamps along the sides of the sequencing apparatus Tighten the screw knobs firmly but do not over tighten 4 Verify that the upper buffer chamber drain valve is closed and fill the upper buffer chamber with approximately 450 ml of electrophoresis buffer Make sure no buffer is leaking from the upper buffer chamber Fill the front chamber of the lower buffer tray with approximately 450 ml of electrophoresis buffer Be sure no bubbles obstruct buffer contact with the lower edge of the gel Note Do not overfill the buffer chambers or allow buffer to make contact with the electrode mounting nuts Warning No buffer should leak through or around the silicone gasket or down the side of the gel assembly Leakage may allow the upper buffer chamber to drain dry or cause sparking and damage to the apparatus See Chapter 3 Troubleshooting for further information 5 Close the upper and lower safety lids and connect the DC power cord
24. m Weight 6 32 kg Gel Dimensions 31x38 5 cm Maximum gel thickness 1 6 mm Voltage amp Current Range 2 000 VDC Max at 0 125 mA 0 5 A Max Electrode material Pt Ti wire Operating Temperature Range 4 30 C non condensing atmosphere Construction Flame retardant ABS acrylic aluminum silicone 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 SPM 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 occurred 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 Decontamina
25. mbs 28 cm Mylar Sharkstooth Comb 28 cm Vinyl Sharkstooth Combs Glass Plates pair of short and long Description 0 19 mm thick Mylar 0 35 mm thick Mylar 0 4 mm thick 0 8 mm thick 1 6 mm thick 0 4 mm thick 0 8 mm thick 1 2 mm thick 1 6 mm thick Complete with two 0 4 to 1 2 mm thick side spacers and 12 foam blocks Package of 12 0 4 mm thick 0 8 mm thick 1 6 mm thick 0 4 mm thick 0 8 mm thick 1 6 mm thick 0 4 mm thick 0 8 mm thick 1 6 mm thick 25 pt 0 19 mm thick pkg of 6 25 pt 0 35 mm thick pkg of 6 25 pt 0 4 mm thick pkg of 6 49 pt 0 4 mm thick pkg of 4 62 pt 0 35 mm thick pkg of 2 50 pt 0 4 mm thick pkg of 2 Precision flat clean edges Long 333 mm 13 12 wide x 419 mm 16 50 long Short 333 mm 13 12 wide x 394 mm 15 50 long S2 Casting Clamp Package of 1 Catalog 21105309 21105366 21108014 31109010 31109028 21105077 21105069 21105325 21105051 21107016 21965017 21035043 21035050 21035068 21035076 21035084 21035092 21035100 21035118 21035126 21105317 21105341 21045018 21045026 21035134 21046016 11034014 21105432 Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com HV Gel Sealing Tape 1 5 in x 72 yd 1 roll 10 rolls Replacement Part Power Cord Replacements 1 black amp 1 red 122 cm long Silicone Gasket grey S2 Upper Buffer Pt Ti Electrode R
26. ng instructions for the Model S2 Apparatus 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 DANGER HIGH VOLTAGE This system requires a 1 000 to 3 000 VDC power supply for operation and is therefore a source of high voltage The power supply should have earth ground leakage protection and open circuit sensing Although equipped with a safety interlock system this equipment must always be operated with extreme caution Careless handling can result in possibly fatal electrical shock 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 Certain reagents indicated for use in this manual are of a hazardous nature The researcher is cautioned to exercise care when handling these reagents Equipment used in these procedures e g high voltage power supplies should be used following the manufacturer s safety recommendations e Always follow the power supply manufacturer s recommendations for use and follow safety procedures e Never operate damaged or leaking equipment Inspect the a
27. ntly Force water or buffer through the drain outlet with a syringe or pipette to remove possible dried buffer residue airlock or collapse in the drain tubing Wiggly or slanting bands bands are not Verify that the wells are free of particles and bubbles straight lines or parallel to the top edges of before and after loading samples the gel 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 casting All bands appear as doublets each band is Concentrate the sample and use a thin 2 to 3 mm represented twice within the same lane gel with a thin 1 mm comb Prevent gel movement during photography The gel dye front is not straight Verify that the system is level Make sure the system is not in a draft or otherwise being cooled unevenly Verify that the buffer depth in both buffer trays is sufficient to provide complete even contact across the full width of the gel Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Buffer leaks from the upper buffer chamber Sparks or burn marks appear at edges of aluminum plate Verify that both electrode carriers are properly clipped in place Make sure that the spring clips are placed over the side spacers Distortion of the gel thickness will cause the dye front to run at
28. o 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 32P 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 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Once the unit has been determined to be radiation free 100dpm cm remove all the hazardous and radioactive labels from the unit If the
29. oam blocks may be replaced as needed using the extras provided with the Model S2 Apparatus Seal the sides and bottom of the assembled glass plate sandwich with gel sealing tape as shown in Figure 2 Tape the sides first a b and then the bottom c Extend the tape around the bottom corners in both directions to ensure an adequate seal Apply the tape as smoothly as possible to avoid forming air channels or bubbles along the edges of the glass plates Rub the tape firmly onto glass to eliminate air channels and ensure a liquid tight seal Place two accessory spring clips available separately along each side near the middle and bottom of the glass plate sandwich This will help maintain uniform gel thickness while Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com Figure 2 Taping Glass Plates pouring the gel It is important for the spring clips to be placed over the side spacers only Clamping unsupported glass will distort the thickness of the gel TOR 2 1 2 PREPARATION OF GLASS PLATES USING THE S2 CASTING CLAMP The S2 Casting Clamp can be used in place of the gel sealing tape and spring clips to seal the edges of the glass plates while casting the gel Assemble the glass plates as before and place the assembled sandwich in the casting clamp following the instructions below 1 Make sure that the inner surface of the casting clamp is clean of all dried acrylamide an
30. ogether while taping the gel Make sure glass plates are seated flat in the electrophoresis unit Make sure the gel electrophoresis unit is level 4 0 REFERENCES Anderson S 1981 Nucl Acids Res 9 3015 Bankier A T Weston K M and Barrell B G 1987 Methods Enzymol 155 51 Life Technologies Inc 1985 M13 Cloning Dideoxy Sequencing Instruction Manual Blakesley R 1983 Focus 5 1 1 Kuebbing D 1983 Focus 5 2 1 Martin R 1987 Focus 9 1 8 Maxam A M and Gilbert W 1980 Methods Enzymol 65 499 Sanger F Nicklen S and Coulson A R 1977 Proc Nat Acad Sci USA 74 5643 1 2 3 4 5 Johnson Dow L Mardis E Heiner C and Roe B A 1987 BioTechniques 5 754 6 7 8 9 10 Smith A J H 1980 Methods Enzymol 65 560 11 Tullius T C Dombroski B A Churchill M E A and Kam L 1987 Methods Enzymol 155 537 12 Hartley J and Xu L 1994 Focus 16 52 Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 5 0 RELATED PRODUCTS AND REPLACEMENT PARTS Accessory Spacer Sets Includes two side spacers and 12 foam blocks Individual Bottom Spacers Wedge Spacer Set Silicone Spacer Foam Blocks 16 tooth Machined Analytical DELRIN Comb 20 tooth Machined Analytical DELRINComb 32 tooth Machined Analytical DELRINComb 14 cm Mylar Sharkstooth Comb 14 cm Vinyl Sharkstooth Comb 14 cm Vinyl Double fine Sharkstooth Co
31. pparatus electrical connections and power cords prior to use e For maximum safety always operate this system in an isolated low traffic area not accessible to unauthorized personnel e Before applying DC power to this equipment make sure that all electrical connections are secure and that power cords show no signs of damage e Always turn off the DC power source before disconnecting DC power cords from the sequencing apparatus Disconnect power cords from the power source first and then from the apparatus Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 1 3 COMPONENTS The Model S2 Apparatus is designed for easy assembly and reliable performance Please refer to Figure 1 to identify the following principal features and components e Electrophoresis apparatus with upper buffer chamber buffer chamber drain valve silicone gasket removable lower buffer tray integral gel clamps and safety lids e One pair of 122 cm 48 red and black power cords e One pair of glass plates one short one long Long 333 mm 13 12 wide x 419 mm 16 50 long Short 333 mm 13 12 wide x 394 mm 15 50 long e One pair of 0 40 mm thick vinyl side spacers e One pair of 0 35 mm thick Mylar side spacers e Package of four 0 40 mm thick vinyl sharkstooth combs e Package of two 0 35 mm thick Mylar sharkstooth combs e Package of 12 adhesive backed foam blocks for side spacers e One roll of high voltag
32. s to the sequencing apparatus and the DC power supply Warning All banana plug connections must be fully seated to prevent potential sparking and fire hazards 6 Before loading samples pre electrophorese a gel for 15 to 45 min see Table 2 for recommended DC power settings for various gel thicknesses Best results are achieved with a gel surface temperature of 50 C Warning Excessive power will cause the gel to overheat and crack the glass plates Table 2 Recommended Electrophoresis Power Settings for Different Gel Thicknesses Gel Thickness mm Watts W V x A Volts V Current mA 0 2 2 200 to 2 600 20 to 35 55 0 6 1 500 to 1 900 30 to 45 65 4 0 0 8 1 100 to 1 200 60 to 90 0 1 6 7 600 to 700 95 to 125 Wedge 85 to 90 1 200 770 Note Power conditions were determined for a 50 C surface temperature with 1x TBE buffer 70 4 to 1 2 mm thick Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 2 2 2 LOADING SAMPLES 1 Prepare the samples by heating in an appropriate loading buffer to a temperature of 90 C to 100 C for 3 to 5 min and then chilling on ice 2 At the end of the pre electrophoresis period turn off the power supply and disconnect both DC power cords first from the power supply and then from the sequencing apparatus before opening the upper safety lid 3 Immediately prior to loading the samples rinse the wells of the gel with electrophoresis buffer Use a
33. tion 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 with 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 gre
34. uffer rinse both chambers of the tray thoroughly with deionized water and place the tray back in the sequencing unit Disassemble the gel sandwich by prying off the short or siliconized glass plate working gently from a bottom corner with a thin spatula For autoradiography transfer the gel to a solid backing such as paper or used film Rinse combs side spacers and glass plates to remove buffer and gel fragments and dry all components before storing Apogee Electrophoresis 101 Kane Street Baltimore MD 21224 USA apogeephoresis com 3 0 TROUBLESHOOTING GUIDE Some suggestions for resolving 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 SUGGESTED SOLUTION Gel Solution leaks from the bottom Lower the angle of the glass plates when pouring the gel during casting Check that the inner surface of the casting clamp is clean and free of debris and grease Make sure that the glass plates are firmly seated against the inner surface of the casting clamp The glass plates crack The gel is too hot Use a lower wattage or voltage setting The upper buffer chamber will not drain Check that the valve knob has been released sufficie
35. ult the Material Safety Data Sheet for more information Table 1 Approximate Gel Solution Volume Requirement for Different Gel Thicknesses Gel Thickness mm Volume Required ml Wedge 0 4 to 1 2 mm thick 0 2 35 0 4 65 0 8 130 1 6 260 i 140 2 Usea large syringe squirt bottle funnel or beaker to pour the gel solution between the glass plates Hold the assembled plate sandwich at a 45 angle on one bottom corner so that the gel solution flows evenly down along the lower side spacer Maintain a constant even flow to reduce the chance of forming bubbles in the solution 3 As the gel sandwich fills gradually lower the glass plates until they rest on the bottom edge approximately at a 5 angle from the bench The gel mold should be slightly overfilled to ensure complete polymerization at the top Note If a bubble forms while the gel is being poured raise the glass plates into a vertical position and either tip the gel solution away from the bubble or carefully tap the plate sandwich at the bubble to make it rise to the surface Once all bubbles have been removed return the glass plates to their previous position 4 Before the acrylamide polymerizes insert a well forming comb into the top of the gel If you are using a sharkstooth comb insert the flat edge of the comb between the glass plates to a depth of 2 to 3 mm below the top of the short glass plate Figure 4 Keep the flat edge shallow to simplify subsequent
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