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WormPCRPickonewormandplaceitina2.5ldropoflysisbufferinthecapofaPCRtube.Closeandcentrifugebrieflytomovetothebottomofthetube.Freezethetubesat-70°Cfor15min.Theideaistodoafreeze-cracktohelpliberatetheDNA.Checkthatthesolutionactuallyfreezes.Overlaywithadropofmineraloilandincubateat60°Cfor60minutes,followedby95°Cfor15minutes.Coolto4°C.Pipette22.5lofPCRmastermixontothetopofthemineraloiloverlay.Setsampleinicebucketuntilallsampleshavebeenprepared.Microfugebrieflytomovethemastermixthroughthemineraloiloverlay.Rapidlyheatthesamplesto94°Candcycle30timesthroughaprogramappropriateforthetemplateDNAandtheprimersyouareusinginthereaction:Meltingstep:94°Cfor30sec(standard)Annealingstep:~5°Cbelowmeltingpt.oftheprimersandusuallyheldforbetween30secondsand1min.Extensionstep:72°Cstandardtemp.andest.timebasedonlengthoftemplate(approx.1minper1kb)Analyze10mlofeachsampleona3.0%Metaphoragarosegel,ora6%acrylamidegel.BesuretorunStyIorsomeothermolecularweightMarker.Lysisbuffer(storeonthebenchtopwithoutproteinaseK).60g/mlproteinaseK10mMTris-Cl,pH8.250mMKCl2.5mMMgCl20.45%Tween-200.05%gelatinMastermix.Prepareamastermixcontainingthefollowingamountsofeachcomponentperreaction.Makeenoughmastermixfor(n+1)reactions.2.5l10Xamplificationbuffer2.5l2mMdNTPs1.5l25mMMgCl20.12l5U/lTaqpolymerase(0.6Units/reaction)1.25lprimermix(20pMol/lconc.ofprimercombination)14.63ldH2O