MATERIALS:

	2-glassplates
	1sharks-toothcombandspacers
	Whatman3mmpaper
	30or40%acrylamide-bis(19:1)
	10XTBE
	urea
	10%ammoniumpersulfate
	TEMED
	60cc.syringe
	Rain-ex

Preparationofglassplates:

Washandrinsewellthesequencingplates.Silanizethesmallernotchedplateasfollows.ApplyasmallamountofRain-extotheinsidesurfaceofthesmallplatewithaKimwipe.Letairdrytoathinfilm.Rinsewellwithdistilledwateranddry.Anadditionalcleaningwithaglasscleanerishelpful.CleananddrytheThermoplateandplaceoverbenchwrappaper,elevatedbutlevel.Layspacersalongthesidesevenwiththetopandbottomoftheplate.Placenotchedplate,silanized-sidedown,ontopoflargerplateandclamp.

Preparationofgelsolutionfor100mlofan6%8Mureagel:

	15mlof40%acrylamide-bis(19:1)
	10mlof10XTBE
	46gurea
	~30mlwater

Placeinabeakerwithastirbarandplacethisbeakerinsidealargerbeakercontainingsomewarmwater.Stiruntildissolved.Donotheat.Measurevolumeandaddwaterto100ml.Add666μl10%ammoniumpersulfateand40μlTEMEDandstirgently.Bringupsolutionina60cc.syringeandletsolutionflowdowncenterofnotchedplatebeingcarefulnottogenerateanybubbles.Whenplatesarefilled,placeflat-edgeofsharktoothcombinafewmm-notalltheway.Clampthetopofthegeltoholdthecombandallowthegeltopolymerize(1-2hr).GelscanbepreparedinadvanceifthetopofthegeliswrappedinSaranWrap.

RUNNINGSEQUENCINGGELS

Torun,set-upthegelintheelectrophoresischamberbeforeremovingthecomb.Add1XTBEbuffertotheupperchamberandgentlyremovecomb.Immediatelyrinseunpolymerizedacrylamideandureafromthecombarea.Repeatthisagainbeforeyouloadyoursamples.Addbuffertothebottomchamberonlywhenyouareconfidentthattheupperchamberisnotleaking.Prerunthegel35-50wattsfor30-60min.Thegelshouldbewarmtothetouchandthesamplesshouldbeloadedontoawarmgel.Toloadsamples,turnoffpower,rinsecombarea,andplacesharkstoothcombssuchthatthetipofthecombjustmeetsthetipofthegel.Loadpreheatedsamplesbetweenthetips.Ifanumberoftemplatesarebeingrun,youcanavoidtoomuchdiffusionofyoursamplesintoadjacentlanesbyrunningtheDNAfor1or2samplesintothegelbeforeloADIngthenextset.Runthegelat35-40wattsuntilthebromophenolbluegetsclosetothebottom.Todoubleload,addadditionalsampleatthistimeandrunthegeluntilthebromophenolbluegetsclosetothebottom.Disassembletheplatescarefullymakingsurethegelstickstothebottomplate.Addfixativesolutiontogelandletsitfor>15min.DrainexcessfixativesolutionoffofthegelandplaceapieceofWhatmanpaperonthegelandpeeloff.DrythegelthoroughlyonageldryerwithSaranWrapontopofgelonly.Dryfor30-90minat80EC.RemoveSaranWrapbeforeexposingthefilm.(If,bychance,thefilmshouldsticktothegelwhenitistimetoexposeit,DONOTPUTTHEFILMINTHEPROCESSOR.NOTIFYTHEINSTRUCTOR).(For³²Pgels,aSaran-Wrapped-wetgelcanbeexposedtofilmwithoutfixingordrying).For³⁵Sgels,exposefor20-24hr.

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