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P81 Single Sided Vertical Electrophoresis System User Manual
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1. Reagent Information RUNNING BUFFER TGS Tris 3 0285g L Glycine 14 4g L SDS 1 0g L pH 8 3 Laemmli 1970 q s to IL Note For Native Protein Electrophoresis do not add SDS A Table 3 1 Sample Buffer 2X Concentration Final Concentration 250mM 6 057g 06069 125mM 0 002 Bromo Phenol Blue 0002g 0 001 add sample buffer 1 1 with sample solution Caution 2X Sample Buffer containing 2 mercaptoethanol should be prepared in a fume hood 0 2M final concentration Dithiothreitol DTT may be used in place of 2 mercaptoethanol DDT should be added before use and made fresh A Ac rylam ide Solution stock acrylamide solution for Table 3 2 29 2g Acrylamide and 8g bis Acrylamide q s 100mL H2O Table 3 2 Gel Preparation SDS Page continuous buffer system Acrylamide gr 05M Sodium Phosphate Butter p72 50 00 D 185 WAT 08 E EE E TEMED 0 015 0 015 0 015 0 015 0 015 iss 1 me The columns represent volumes ml of stock solutions required to prepare 30ml of gel mixture 32 Vertical Electrophoresis System Thermo Scientific Section 3 Technical Tips Combs STANDARD 0 5mm A 0 8mm C and 1 5mm D thicknesses Standard Preparative One long well and one marker lane CUSTOM COMBS Call Technical Services for more information Notched Alumina Notched Alumina plates can take the place of the notched glass when PI ates for P8DS Only casting and allows for better heat exchange than
2. 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 Vertical Electrophoresis System Thermo Scientific Thermo Scientific Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Table of Contents General Information 1 1 Unpack and Check Your Order 422 NEE tx eek wwe 1 1
3. CASTER The Multiple Gradient Caster System features an easy to use casting base and specially designed spacer plates for quick casting of high quality linear or gradient gels Using the acrylic and foam spacer plates allows you to cast from one to five gels simultaneously A silicone gasket provides a leak proof seal and the casting port allows the casting of gradient gels from the base of the caster Rabbit cres ett Sek rodea ears P7 CST Gel Size 00 eee eee eee 10cmWX 10cmL GEL CASTER The patented Gel Casting System provides a simple method of casting acrylamide gels without taping or special sealing of the gel plates Plastic pouches hold W _ A glass plates and spacers snugly together in the casting stand while pouring These pouches meet specific measurement tolerances to allow for a tight fit around glass plates Gels may be cast ahead of time and sealed inside the plastic pouch creating your own pre cast gels Up to four gels may be cast at one time POC eiua o SUR esaet DE ard Rd JGC 4 Gel Size ut ERRARE 10cmWX 10cmL RELATED PRODUCTS Panther Semi dry Electroblotter Puffin Single Sided Vertical Gel Electrophoresis System Bandit Tank Style Electroblotter Powdered Buffers Silver Stain Pro Blue Vertical Electrophoresis System 6 3 Section 7 Warranty Information ENENSDEL 1006 OSI cL 6 0 noa uoneuuojul AJUCIIEM JO JOJNQUISIP 290 Jno joe4juoo YSN eui episino su
4. supply 2 Remove the lid Slide and lift the upper buffer chamber from the lower buffer chamber and drain buffer chambers separately 3 Loosen wing knobs and slide side clamps to remove gel cassettes The gel is ready for staining and blotting Contact Technical Services or see the product catalog for additional staining and blotting accessories 2 2 Vertical Electrophoresis System Thermo Scientific Glass Spacers Thermo Scientific Section3 Technical Tips BLANK The plate which faces you during electrophoresis All gel sandwiches require one piece of blank glass NOTCHED The plate which faces the chamber during elec trophoresis Spacers are placed over the ears of the plate when casting vertical gels Buffer accesses the gel between the ears OFFSET Offset plates may be used in place of notched plates They require sponge tips mounted on the spacers Sponge tips take the place of the ears and prevent buffer from running out of the upper buffer chamber from the sides FROSTED a BLANK n NOTCHED OFFSET Frosted plates are used for vertical agarose electrophoresis One side of the plate has a rough surface to prevent agarose from sliding down STANDARD Protein spacer sets include two side spacers and one bottom spacer Spacers and combs must be of identical thickness to be used together SPACER Sa SPONGE TIP Vertical Electrophoresis System 3 1 Section 3 Technical Tips
5. uonejjeisu oqe Buipnjour esuedxe s ouueu Je peoejdoJ 9q IM d ysuewy Jom JO elasjeuJ ui Buruuojuoo uou eq oj ue oJd sued jueuoduJoo sujuouu 9c XIS UIU 38114 ayy BUNG Jeuwo juanbasqns Aue o spuajxe uonoajold AJUeWEM ay palanijap si juaudinba noA au eujes y A oyeuuixoJdde ye Pays OUI OB Wu jueJem ay os aul Buiddius smojje siu AuIpDEL uno woy peddius si jueuudinbe snoA ep ou WO SY M OM SUB S polla g AueJeM SUL VSN ALNVYYVM SLONGOYd IMO OISILNAISS 43HSIA OWYSHL 7 1 Vertical Electrophoresis System Thermo Scientific Section 7 Warranty Information ERENODEL 1006 OSI cL 6 0 es uoneuuoJul AJUBIIEM JO JOINGLISIP 290 Jno J98 1JUO9 YSN eui episino suoneordde jeioeds pue s s eoueuejureuJ uoneJedo AjueJeM Juswd nba uo suonsenb noA Jamsue 0 peal 9J 9M EILF EJE OVZ L Jo epeueyd Jo VSN 158y 8 py 008 1 1e 1ueungedeq samas eoiuuooe JNO jeo aseajd peJinbaJ s eoiues 1ueuudinbo y eoueuejureui eAgueAeJd pue uonejedo uonejejsur jueuudinboe 1ejep Anal sjenueuJ uononuajsui paud SOALIE jueuudinbe noA aJojaq uoneuuojul uoneJedeud ous SAISUBYSJGLUOD UM dau 0 Apes si a914O sejes OUSOU 890 INOA s onpoud Jo sso Jo syyoJd 1so 0 sobewep uonejyuui moyjm Buipnjour sebewep jenuenbesuoo Jo Depu Aue 104 jqe eq jou eys oway AlddV TIVHS ISOdANd YVINOILYVd V HO SSANLIS HO ALITISVLNVHOUYFN JO S3LENVSHSHVM ON AFNIANI YO WHO N3 LLIHM YSHLSHM S3ILNVHSHVM H3HLO TIV 30
6. NA NI ANY FAISNTIXF SI ALNVHSHVM SIHL uoneunsep go peddius sue sued 1ueujeoe doJ pue pied aBejsod oway o peuunjeJ eq Jsnw sued BuriuJojuoo uou UE uodo s ouueu 1v 3ueuudinbe Jo 3jueuoduJoo Aue jo uJnjeJ Joy jenoJdde sod SAID 1snuu jueunjedoeq searues eoiuuo9 SU poled jueJem eulbiio ou puo eq Led juauoduloo y 0 Jo Juswd nba y JOYS 0 JueJem OU PUS X9 jou eys AUeJJeM siy Jepun Juswd nba Jo sued Jusuodwos Jo edau JO jusuusde doy jJU amp JIeM siy wo pepnjoxe ale sjexseb pue sia ssej6 sway ejqepuedx3 suredeJ Aue jo eoueuuo Jod 0 JOUd uonoeJip pue uoneuiuuJejep jueJeM JO pejoejuoo eq snu jueunjedeq sasas eoluyos SU 1ueuleeJ6e AUBJJEM siy Aq pajaros jou SI uoe pue uoneJqijeo uonejeysu oqe Bundeoxe esuadxa s ouueu Je peoejdoiJ 99 WAN diusueunjJoM Jo IEUSIEU ui BuiuuJojuoo uou aq oj ue oJd sued 1ueuodujioo sujuoui 9e xis ALU 15414 OU Bung Jaumo juenbesqns Aue o spuajxe uoi 98 01d juBJJEM OU peJeAr op si 3jueuudinboe noA au Wes y AjojeuurxoJdde Je Pays out OB WA AjueJJe ay os eui Buiddius smojje siu Apioey ano Wo peddius si Juswd nba noA opp eui OU syjuow OM SUEIS pollag Aueuemn SUL TWNOLLVNYSLNI ALNVSHVM SLINAOUAd IMO OISILNAIOS JIHSII ONYFHL Thermo Scientific Vertical Electrophoresis System 7 2 Thermo Fisher Scientific 401 Millcreek Road Marietta Ohio 45750 United States www thermofisher com ThermoFisher SCIENTIFIC
7. Single Sided Vertical Electrophoresis System P81 Operating and Maitnenance Manual 7007341 Rev 0 Visit us online to register your warranty www thermoscientific com warranty o Owl Preface MANUAL NUMBER 7007341 0 5 3 12 Transfer to Marietta was The Puffin 11 2002 REV ECR ECN DATE DESCRIPTION By Thermo Scientific Vertical Electrophoresis System i Preface CAUTION Contains Parts and Assemblies Susceptible to Damage by Electrostatic 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 Running conditions for this unit should not exceed the name plate readings found on the lower buffer chamber Do not move the unit unless the power source to the unit has been disconnected A Statement of Proper Use Use this product only for its intended purpose as described in this manual Do not use this product if the power leads are damaged or if any of its surfaces are cracked This Owl System is designed to meet IEC 1010 1 safety standards IEC 1010 1 is an internatio
8. Using the System el 2 1 Rumninethe Gael 425 2 Ya wars nes 2 1 After the el RUN uoto sv edo S REBATE ORE 2 2 Running Conditions 4425644 eee Se ee ey Ge ea ee a 2 2 Technical Tips 2 aa 3 1 GCs ege gE A ator N E Fe tee ner Ee 3 1 Spaces Au eit A wp up udo x e e aaa e ege e RN 3 1 Acrylamide SOLON eso nO sao o e RN 3 2 Reagent Information sessio aie E vege etek re EXP Ee 3 2 Offset vs Notched Glass Maura oor VLA uid cs oiu Corda 3 3 Notched Alumina Plates for PSDS Onk 2222220 3 3 Cobain ai Me tU ICM O e A e aS 3 3 Troubleshooting ug geed xem ee rn e Su 4 1 Care and Cleaning cece cece eee eect eee eeeeeeeeees 5 1 Optional Equipment cece eee eee eee eee eee 6 1 Vertical Electrophoresis System V Section 1 General Information The Single Sided Vertical Electrophoresis System is simple rugged and provides excellent results The upper buffer chamber UBC of the single sided vertical system extends the length of the gel to distribute heat evenly across the entire gel surface Side clamps press glass plates against asilicome gasket to provide univlorm pressure and form a leak proof seal This heat and pressure distribution system includes a notched alumina ceramic plate which may be used in front of the notched glass for cooler faster runs Unpack and Check Before starting unpack the unit and inventory your order If any parts are Your Order missing contact Technical Services immediately Referen
9. and check the setup of the appa ratus to ensure a secure seal with gasket Streaked bands Overloading of sample Use less protein or sample when loading Centrifuge sample before adding sample buffer or use a lower acrylamide gel Skewed bands Gel has not polymerized properly at wells The upper buffer chamber is leaking either through the gel or along the sides Sample has precipitated Do not move spacers after polymerization and make sure that gasket is seated firmly against the glass Always load your samples with an empty lower buffer chamber so that leaks are caught before you begin the run Leakage of buffer along sides or along EES spacers inside the gel assembly If using a reducing agent prepare fresh sample buffer every 30 days Increase the concentration of 2 mercaptoethanol or dithiothreitol in sample Due to reoxidation or insufficient reduction Double bands doublets file sample Use glass appropriate for the unit If this is not possible use an extra piece of blank glass to Gel is too thin for the clamping system take up the space If the clamps are used with their flat side against the glass thinner glass may often be used Glass cracks when putting gel assembly in unit Wash gasket after each use to remove salts If gasket is old and has lost its flexibility it may Gasket is old or flattened making it impossi need to be replaced If unit has been previously ble to make a good seal overtig
10. ce the order or catalog number on your invoice and check the corresponding parts list Table 1 1 Parts List armen Iess eppes E Thermo Scientific Vertical Electrophoresis System 1 1 Section 1 General Information 12 Vertical Electrophoresis System Table 1 2 Specifications Unit Model Number uuu ees 10cmW x 10cmL amp 10cmW x 8cmL Power Supply Leads Comb 2 Notched Alumina Plate Notched Glass Upper Buffer Chamber UBC di Blank Glass Spacer Set Lower Buffer Chamber LBC Figure 1 1 Exploded Parts Diagram Thermo Scientific Running the Gel Thermo Scientific Section 2 Using the System Note Owl offers a multiple gradient caster and a gel caster See Section 6 for details 1 After the gel is cast place the gel with the notched or offset glass plate facing the inside of the upper buffer chamber The gel cassette must be placed squarely on the corners in order to provide a good seal with the gasket and avoid leakage of buffer from the upper buffer chamber to the lower buffer chamber 2 Add running buffer to the upper buffer chamber making sure the running buffer is 3mm below the top of the blank glass ensuring sufficient contact with the top of the gel surface Be sure that the running buffer is not leaking from the upper buffer chamber to the lower buffer chamber If buffer is leaking you will need to drain the UBC and reset the cassettes 3 Remove combs by gently pullin
11. centrated Diffusion of sample during run in stacking Increase of stacking gel or increase current gel by 2596 when stacking Match the ionic strength of the sample with L ionic strength of ower ionic strength of sample bett gal lonic strength of sample is higher than that Desalt the sample or use sample buffer of Bands are narrower than sample wells p of the gel the same strength as the gel 4 2 Vertical Electrophoresis System Thermo Scientific Section5 Care and Cleaning A few tips about caring for your system follow Caution Organic solvents cause acrylic to craze or crack Clean all acrylic systems with warm water and a mild detergent Do not use ethanol or other organic solvents to clean these products Do not autoclave bake or microwave your unit Temperatures over 50 C can damage the acrylic A 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 diluted 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 A To order RNase AWAY conta
12. ct Technical Services Part Number oua cm vp E ord as Description A aae e yr S Ed 250ml botte 7002 rita 475ml spray bottle TODS BEA ae oM A N 1 liter bottle 7 008 da da oe RA 4 liter bottle Rnase AWAY is a registered trademark of Molecular BioProducts Thermo Scientific Vertical Electrophoresis System 5 1 Section 5 Care and Cleaning Care of Acrylic The following chemical compatibility chart is supplied for the convenience of our customers Although acrylic is compatible with most solvents and solutions found in the biochemical laboratory some solvents can cause substantial damage Keep this chart handy to avoid harm to your apparatus by the use of an inappropriate solvent Codes S Safe no effect except possibly some staining A Attacked slight attack by or absorption of the liquid slight crazing or swelling but acrylic has retained most of its strength U Unsatisfactory softened swollen slowly dissolved D Dissolved in seven days or less 5 2 Vertical Electrophoresis System Thermo Scientific Section 5 Care and Cleaning Table 6 1 Chemical Compatibility for Acrylic Based Products Soma Fe Wem EP Ee P Rss EP IN e em EP Fam ja em O ET E pent H es O Reese NE 5 fm O EC EN TEN EN Bats Ehesea o emer je mw fo E u NN EN Dune Te CA CE Desa O emnes CTN e eo CTN NN ESOO EN E u CON E Ee ac s CEIC LA EC EEC umen DI CN Dem CON CEI fs Ta LN Bem p O O This list does not include a
13. g straight up from the gel Carefully load samples into the wells formed by the comb Rinse wells with water 4 Add buffer to the lower buffer chamber to approximately 2 3mm above the base of the gel using the fill line as a guide max fill The bottom end of the gel agarose assembly should be in contact with the running buffer 5 Set the safety lid onto the unit so that the power supply leads are connected Begin the gel run Table 2 1 Maximum Buffer Volumes Unit vs hed Biken P81 Upper Buffer Chamber siones 100ml Lower Buffer Chamber 50ml Vertical Electrophoresis System 2 1 Section 2 Using the System Runnin g Conditions Running conditions depend on several parameters e Buffer system used e Whether or not heating would affect subsequent processing of the proteins or gel e Thickness of the gel e How fast the gel will be run for example set it up in the late afternoon and have the gel done the next morning or have it done in 45 minutes or less A guideline for 2nd dimension gels the range would be 30 80mA constant current Example For an SDS PAGE gel in the P8DS that is 0 8mm thick and temperatures over 37 C are not an issue 40mA per gel is appropriate If the gel were 1 5mm thick the setting could be 60mA or higher Table 2 2 Recommended Running Conditions UN use P81 Constant Current sos kode ee et 15 35mA gel Time Requirements ve ed eee 30 90 minutes After the Gel Run 1 tumor power
14. glass This is important when the protein is heat sensitive or if a gel needs to be run a little faster without the negative effects of heating Heating the gel during the run can cause smiling and other distortions of the gel Offset vs Notched _ 4i units require a blank piece of glass and an SPONGE TIP Glass offset or notched piece of glass Offset glass is glass that is about 2cm shorter than the blank piece without ears on the sides Notched glass has two SPACER ears that are left behind when a cut is made in the middle of the top of the glass Both offset glass and notched glass allow the gel and samples to make contact with the upper buffer chamber Offset glass has to be used with sponge tips which take the place of the notches on the glass The advantage of offset glass is that this glass is more rigid Notched glass is easier to use and does not require the addition of sponge tips Thermo Scientific Vertical Electrophoresis System 3 3 Thermo Scientific Section 4 Troubleshooting Problem Will occur when adjoining lanes are loaded Broad lanes at bottom of gel mass with dissimilar samples Normal in gradient gels Degas gel solution before casting and increase APS and TEMED concentrations The comb can be wiped with TEMED just prior to casting to improve polymerization Salt concentration is too high in sample Dialyze sample or use desalting column Check gel to make sure that it is a solid slab inside the glass
15. htened the gasket may need to be removed and reseated A cracked and dry gel often is an indicator of overheating Vertical Electrophoresis System 4 1 Section 4 Troubleshooting Problem continued Cause continued Solution continued Check buffer recipe remake buffer and try again See if voltage produced by the Longer run time Buffer is too diluted current you are running at is the same If it differs significantly your buffer may not have been made up correctly Make sure that the gel assembly is seated firmly against the gasket Remove gasket wash in warm water to remove excess salts and place the gasket back in the groove If the clamps have been overtightened in the past the gasket can be pushed too far into the gasket groove and will not make a seal Upper buffer chamber is leaking Use running conditions as stated in table Running at too low a current 3 2 When running at constant current the current value is per gel Check buffer recipe remake and try again If voltage is lower than usual when running at constant current the buffer is probably too dilute Lo Voltage or current set too high Turn down current setting Smiling of dye front Center of gel is running hotter than ends Turn down current setting Make sure that the samples are loaded Bands spreading outward Diffusion of sample when loading quickly and the power is applied as soon as possible after loading Running too fast Buffers are too con
16. ll possible chemical incompatibilities and safe compounds Acrylic products should be cleaned with warm water a mild detergent such as Alconox and can also be exposed to a mild bleach solution 10 1 In addition RNAse removal products are also safe for acrylic Contact Technical Services with any questions Thermo Scientific Vertical Electrophoresis System 5 3 Thermo Scientific Section 6 Optional Equipment Contact the Technical Services department to order replacement parts Table 6 1 Replacement Parts Be m E Blank Glass Plates 3 32 thick Notched glass plates 3 32 Thick Frosted Notched glass plates 3 32 Thick Frosted Blank Glass Plates 3 32 Thick Notched Alumina Plates 1 0mm Thick Spacers 0 5m Spacers 0 8m Spacers 1 5m m Thick m Thick m Thick Blocking Plate for Single Gel Operation Spacer Placer pkg of 3 P7 10G 10cmW x 10cmL P7 10R 10cmW x 10cmL P7 1 OFR 10cmW x 10cmL P7 10FG 10cmW x 10cmL P7 10RA 10cmW x 10cmL Vertical Electrophoresis System 6 1 Section 6 Optional Equipment 62 Vertical Electrophoresis System Table 6 2 P81 Comb Options Number Type Teeth Teeth mm Teeth mm Volume LIN PA CSC Ce E E A _ pe qe qe pe E e 1 ese eff Be je m ee er E See Be E mc qm qoe _ Hx mw e Cs efe COC CC Well CCE ilh i EM Thermo Scientific Thermo Scientific Section 6 Optional Equipment MULTIPLE GRADIENT
17. nally accepted electrical safety standard for laboratory instruments 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 ii Vertical 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 Fisher Scientific has contracted with one
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19. 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 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 v Always follow good hygiene practices Y Each individual is responsible for his or her own safety Thermo Scientific Vertical Electrophoresis System iii 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 information about proper setup operation or
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