3B SCIENTIFIC 1012852 [W19925] Owner's manual

Scope of Supply

The model ’Mini’ contains the following items:

• Electrophoresischamberwith corrosionresistantplatinumelectrodes andhigh-quality connectionsforplusand

minuspole

• Safetylidwithattachedpowercords

• 2combs,1.5mmthick,12teetheach

• Instructionmanual

Safety Instructions

• Pleasereadtheinstructionmanualcarefullybeforeusingtheelectrophoresissystem.

• UseonlyCEmarkedpowersupplies.

• Alwaysdisconnecttheelectrophoresischamberfromthepowersupplybeforeremovingthesafetylid.

• Donotputthesafetyliddownonwetsurfaces.

• Alwaysdisconnecttheelectrophoresischamberfromthepowersupplywhennotinuse.

• Thechamberisprovidedforamax.currentandvoltage.Donotexceed150mA,120V.

• Pleasemindthemaximumfillinglevelfortheelectrophoresisbuffer.

Technical Properties

Thehorizontal electrophoresissystem‘Mini’ hasbeen designedin sucha waythatallows theuser tocast andrunthegel

directlyinasinglechamber.Noadditionalcastingequipment,suchasgreaseorsealisrequiredtopreparethegeltrayfor

castingthegel.

Thechamber’sbottomisUV-permeable,thusthegelruncaneasilybedocumentedincaseofusingDNA-fluorescentdyes.Self

evidently,thegelcanalsoberemovedfromthechamberforstaininganddocumentationafterthegelrun.

Please use only UV light with a wave length of 300nm or higher for all applications, as hard UV light (<300 nm) damages

the gel chamber and the nucleic acids.

Ifyouneedonlyshortrunsareneeded,youcanusebothcombswhichthengivestwiceasmanyslotsforsamples.

Warranty

Themanufacturerguarantees thattheelectrophoresischamberwas completelytested beforedeliveryandthatit complies

withtheapplicablesafetyregulations.

Pleasecheckthedeliveryimmediatelyuponreceiptforcompletenessandpossibledamageintransit.Ifanythingisfaultyor

damaged,pleasecontact3BScientificimmediately.

Themanufacturergivesa24-monthwarrantyontheproductconditionalonthechamberbeingusedaccordingtotheinstruc-

tionmanual.Claimsforreplacementorrepairarenotvalidifphysicalabuseisthereasonforthedamage.Anyliabilityfor

conseqentialdamagesresultingfromtheuseoftheelectrophoresischamberisherebyexcluded.Themanufacturer‘sliability

forintentandgrossnegligenceorfordamagesresultingfrominjurytolife,bodyorhealthisunaffectedbythat.

Ourexperienceshows thetwoplatinumelectrodeslast formanyyearsif theproductisused properly. AnyDamageoccur-

ringis usually dueto mechanicalerrors (e.g. bywashing-up brushes)or contactwith so-called“platinum poisons”such

asphosphorus,boronandheavymetals(lead,zinc, etc.). For thisreason,the platinumelectrodesareexcluded fromthe

manufacturer’swarranty.

Inordertointroducedevelopmentspromptly,themanufacturerreservestherighttochangespecificationsandsmallvisual

detailswithoutpriornotice.

General Instructions

Casting of agarose gels

1. Pleasewearprotectiveclothing,gogglesandglovesforheatingtheagarosesolution.

2. Removethesafetylidoftheelectrophoresischambercarefully.

3. Forthepreparationofthegelsolution,onlysuitableagarosesandelectrophoresisbuffersshouldbeused.Forpreparing

theagarosesolution,weighasuitableamountofagaroseinasmallErlenmeyerflask,addanappropriateamountofelec-

trophoresisbufferandastirbar(ifheatingwithheatingstirrers).Notetheweightofthefilledflasksothatanylossduring

heatingcanbecompensatedforbytheadditionofdistilledH2O.Thus,thegelsolutionwillhavetheagaroseconcentration

Electrophoresis Chamber ‘Mini’

English

thatyouneed.Todissolvetheagarose,heattheErlenmeyerflaskeitherinamicrowaveorwithaheatingstirrer.Forthe

latter,usemediumheatingpowerandstirconstantly.Heatinginamicrowaveshouldonlybeforshortperiodsoftime,on

mediumheatinglevel,repeatedmultipletimes.Whenheatinginamicrowave,removetheErlenmeyerflaskfromtimeto

time(remembertoweargloves,gogglesandbecarefultoavoidboiling!)andgentlyswirlthegelsolutioninacircle.Putit

backinthemicrowaveafterwardsandrepeattheentireprocess3-4times,untiltheagaroseiscompletelydissolved.

4. Beforethecastingofthegel,allowthegelsolutiontocooldownto60°C.Castthegelsolutionwithoutanyairbubblesin

thegapbetweenthetwowhiteplasticstripesonthechamber’sground(itscapacityisabout40ml).Theninsertthecomb

(orbothcombsrespectively).Makesurethattherearenoairbubblesontheedgesoftheslots.Gelsmadeofstandardaga-

rosesolidifywithin20minutesatroomtemperature.

5. Afterthesolidificationoftheagarosegel,addsomeoftheelectrophoresisbufferin3-mmlayersinthechamberoverthe

geltopreventifromdryingout.Aftercarefullyremovingthecomb(s),thegelcannowbeloaded,Thecoatedgelcanbe

storedforafewdaysat4°C.Forbestresultsleavethecombinsertedandcoverthegelwithwrappingfilmtoprotectit

fromdryingout.

Loading of gels and electrophoresis

1. Afterthesolidificationoftheagarosegel,coverthegelwiththeelectrophoresisbuffer.Pleasenotethemaximumfilling

levelfortheelectrophoresisbuffer.

2. Removethecomb(s)fromtheagarosegelbygentlymovingbackandforthandthencarefullypullingoutupright.

3. Inthenextstep,theslotsinthegelcanbeloadedwiththesamples.Preparingthesampleswithanappropriategelloading

bufferwhichincreasesthespecificweightofthesamplesmakesiteasiertopourthemintotheslotswithamicropipette

(seesection“gelloadingbuffer”).

4. Whenloadingthegelslots,dipthetipofthemicropipettecarefullyintotheslotandthenslowlypressoutthesample.

Makesurenottodamagethebottomoftheslot.

 Beginnersshouldpractisethiswith„practicesamples“consistingofH2Oandgelloadingbufferonly.

5. AtleastonesizestandardshouldbeappliedoneachgelsothatthesizeofthevariousDNAfragmentscanbedetermined.

6. Nowplacethesafetylidontheelectrophoresischamberandconnectthechambertoasuitablepowersupply.

 Pleasenotethecorrectpolarity.Inanalkalineuptoaneutralmedium,nucleicacidsarechargednegativelyandmigrateto

theanode(redpole).

7. Turnonthepowersupplyandruntheelectrophoresisatavoltageintherange80-120V.Theprocessoftheelectrophore-

siscanbeobservedwiththehelpofthedye,whichisinsidethegelloadingbuffer.

8. Bromophenolblueandxylenecyanolarefrequentlyuseddyesforthegelloadingbuffer.Thesedyesarealsonegatively

chargedandmigrateto theanode.This process,the so-calledco-migrationwiththedouble-stranded DNAfragments,

dependsondifferentfactors,suchasagarosetype,electrophoresisbufferandgelstrength.

 Foraroughestimation:bromophenolbluemigratesin1xTAEelectrophoresisbufferand1%-standardagarosegelinthe

samewayasaDNAfragmentof650basepairs.Underthesameconditions,xylenecyanolmigrateslikeafragmentwith

5000basepairs.

Staining of nucleic acids

Sincethere aredifferentmethods ofstaining nucleicacids, wereferat thispoint tothe correspondingtechnicalliterature

(Sambrooket.al.,1989).

Evaluation / documentation

Sizedeterminationsofnucleicacidfragmentscanbecarriedoutbycomparisonwiththefragmentsofalengthstandard.A

detaileddescriptionofthisprocedurecanbefound,forexample,inMolecularGeneticsbyR.Knippers(2008).

Cleaning and Maintenance

Attention: Disconnecttheelectrophoresischamberfrompowersupplybeforecleaning.

Thegelchamberandthecombsshouldbecleanedwithwarmwaterimmediatelyaftereachuse.Ifnecessary,afewdropsof

awashing-upliquidcanbeadded.Pleasedonotusedishwashingbrushesorthelike,asthiscandamagetheplatinumelec-

trodes.Rinse theelectrophoresischamberaftercleaningwith distilledor demineralisedwatertoprevent theformationof

limescale.

Neverleavethechamberuncleanedforhoursorevenovernightasresiduesfromgelorbuffercancausestainswhicharediffi-

culttoremovecompletely.

Attention:Pleaseavoidthe electricalconnectionsbeing incontactwith water.Ifhoweverthis doesoccur, pleasecarefully

wipedrywithasoftclothandallowtoair-dry.Pleasedonotusepaperbecauseitisoftentooroughandcausessmallscrat-

ches.Donotuseahairdryereither,asthehotaircandamageconnectors,fittingsandtheacrylicmaterial.

Attention:Donotuseethanolnorotherorganicsolventsforcleaning.Itcancausecracks,scratchesorotherunwantedmate-

rialchanges(blindnessetc.).

Required Material and Processes

Electrophoresis buffer

Theelectrophoresisbufferprovides thenecessary ionsforthe electrophoresisprocess. It’sadditionprovidesaconstantpH

valuesothatnucleicacidshavethedesirednetcharge.Nucleicacidsarenegativelychargedinanalkalineuptoneutralmedi-

um.Normally,electrophoresisbuffercontainscomponentsthatprotectnucleicacidsfromdegradation,e.g.EDTA,whichcom-

plexesdivalentcationsandthereforeinhibitsDNases.

Atthispoint,thefrequentlyusedTAEelectrophoresisbufferforelectrophoresisofDNAisdescribedfornon-denaturingcon-

ditions.TAEstandsforTris-acetate-EDTA.Youcaneitherproducethebufferyourselfororderitfrom3BScientificasabuffer

concentrate

Agarose, gel volumes and gel concentrations

Eventhoughvariousagarosesareoffered,thesocalledstandardagaroseiscertainlyofprimeimportance.Forthecastingof

anagarosegelinthischamberabout40mlofgelsolutionisneeded.Pleasepreparealittleextragelsolutiontoallowforthe

factthatsmallresiduesalwaysremaininthevessel.

Optimalseparationofmoleculesofdifferentsizeswillbeachievedbyvaryingtheconcentrationofthegelaccordingtothe

informationinthechartbelow.

Optimal separation ranges of the following DNA length (double-stranded DNA) corresponding to

various concentrations of agarose (standard agarose)

concentration of agarose (%) agarose (g) buffer (ml) optimal separation range (kbp)

 0,5 0,25 50 1–15

 0,7 0,35 50 0,8–10

 1,0 0,5 50 0,5–7

 1,2 0,6 50 0,3–6

 1,5 0,75 50 0,2–4

 2,0 1,0 50 0,1–3

Gel loading buffer

Thesampleswhicharetobeanalyzedneedtobemixedwithasuitablegelloadingbufferbeforeapplyingthemtothegel.Gel

loadingbuffercontainsdye(s)fortrackingtherunoftheelectrophoresisaswellasglycerin,saccharoseandthelike.Therefore,

thepreparedsamplesareheavierthantheelectrophoresisbufferandwilldecreaseslightlyintothegelslotsduringthesam-

pleapplication.Bromophenolblueandxylenecyanolarefrequentlyuseddyesforgelloadingbuffers.Thepatternsoftheir

run(orthesocalledco-migrationtodouble-strandedDNAfragments)dependonthetypeofagarose,gelstrengthandthe

typeofelectrophoresisbuffer.

Foraroughestimation:bromophenolbluemigratesin1xTAEelectrophoresisbufferand1%-standardagarosegelinthesame

wayasaDNAfragmentof650basepairs.Underthesameconditions,xylenecyanolmigrateslikeafragmentwith5000base

pairs.

English

DNA length standard

Forthesizingofthesamples,applyoneDNAsizestandardoneachgelatleasttoonelane.DNAsizemarkesaremadeofDNA

fragmentsoffamiliarsizes.

Staining of nucleic acids

Sincethere aredifferentmethods ofstaining nucleicacids, wereferat thispoint tothe correspondingtechnicalliterature

(Sambrooket.al.,1989).

Declaration of Conformity

Theelectrophoresischamber’Mini’complieswiththeapplicablesecurityrequirementsaccordingtoEN:50081-1(1992)and

EN:50082-1(1992).

Literature

Ausubel,FrederikM.etal.(Ed.),(2005).ShortProtocolsinMolecularBiology,

ACompendiumofMethodsfromCurrentProtocolsinMolecularBiology.Wiley-VCH,Hoboken.

Knippers,R.(2008).MolekulareGenetik.ThiemeVerlag,Stuttgart.

SambrookJ,Fritsch E.F. ManiatisT.(1989). MolecularCloning: ALaboratory Manual.Cold SpringHarbor Laboratory Press,

NewYork.

DNA-Längenstandard

EinDNA-LängenstandardwirdaufjedemGelmindestensineineSpuraufgetragen,umeineGrößenbestimmungderProben

vernehmenzukönnen.DNA-LängenmarkerbestehenausDNA-FragmentenbekannterGröße.

Anfärbung von Nukleinsäuren

Daes verschiedene Möglichkeitender Anfärbung vonNukleinsäurengibt, wirdan dieser Stelleauf dieeinschlägige

Fachliteraturverwiesen(Sambrocket.al.,1989).

Konformitätserklärung

DieElektrophoresekammer „Mini“entspricht dengrundlegendenSicherheitsanforderungengemäß EN:50081-1 (1992)und

EN:50082-1(1992).

Literatur

Ausubel,FrederikM.etal.(Ed.),(2005).ShortProtocolsinMolecularBiology,

ACompendiumofMethodsfromCurrentProtocolsinMolecularBiology.Wiley-VCH,Hoboken.

Knippers,R.(2008).MolekulareGenetik.ThiemeVerlag,Stuttgart.

SambrookJ,Fritsch E.F. ManiatisT.(1989). MolecularCloning: ALaboratory Manual.Cold SpringHarbor Laboratory Press,

NewYork.

Deutsch

Français

L’échelle de marqueur de taille moléculaire

Uneéchelledemarqueurdetailled’ADNestappliquéesurchaquegelaumoinssurunetrace,pourpouvoirmesurerleséchantil-

lons.Cesmarqueursdetailled’ADNconsistentenfragmentsd’ADNdetailleconnue.

Coloration des acides nucléiques

DaesverschiedeneMöglichkeitenderAnfärbungvonNukleinsäurengibt,wirdandieserStelleaufdieeinschlägigeFachliteratur

verwiesen(Sambrocket.al.,1989).

Konformitätserklärung

Commeilyadesméthodesdifférentesdecolorationdesacidesnucléiques,nousattironsvotreattentionsurlalittératurespécia-

liséecorrespondante(Sambrocket.al.,1989).

Littérature

Ausubel,FrederikM.etal.(Ed.),(2005).ShortProtocolsinMolecularBiology,

ACompendiumofMethodsfromCurrentProtocolsinMolecularBiology.Wiley-VCH.

Knippers,R.(2008).MolekulareGenetik,ThiemeVerlag,Stuttgart.

Sambrook,J,Fritsc,E.F.,Maniatis, T. (1989).MolecularCloning: ALaboratory Manual.ColdSpring HarborLaboratory Press,

NewYork.

3B SCIENTIFIC 1012852 [W19925] Owner's manual

Other documents