WormPCR

1. Pickonewormandplaceitina2.5ldropoflysisbufferinthecapofaPCRtube.Closeandcentrifugebrieflytomovetothebottomofthetube.
2. Freezethetubesat-70°Cfor15min.Theideaistodoafreeze-cracktohelpliberatetheDNA.Checkthatthesolutionactuallyfreezes.
3. Overlaywithadropofmineraloilandincubateat60°Cfor60minutes,followedby95°Cfor15minutes.
4. Coolto4°C.Pipette22.5lofPCRmastermixontothetopofthemineraloiloverlay.Setsampleinicebucketuntilallsampleshavebeenprepared.
5. Microfugebrieflytomovethemastermixthroughthemineraloiloverlay.Rapidlyheatthesamplesto94°Candcycle30timesthroughaprogramappropriateforthetemplateDNAandtheprimersyouareusinginthereaction:
   • Meltingstep:94°Cfor30sec(standard)
   • Annealingstep:~5°Cbelowmeltingpt.oftheprimersandusuallyheldforbetween30secondsand1min.
   • Extensionstep:72°Cstandardtemp.andest.timebasedonlengthoftemplate(approx.1minper1kb)
6. Analyze10mlofeachsampleona3.0%Metaphoragarosegel,ora6%acrylamidegel.BesuretorunStyIorsomeothermolecularweightMarker.

Lysisbuffer(storeonthebenchtopwithoutproteinaseK).

• 60g/mlproteinaseK
• 10mMTris-Cl,pH8.2
• 50mMKCl
• 2.5mMMgCl₂
• 0.45%Tween-20
• 0.05%gelatin

Mastermix.Prepareamastermixcontainingthefollowingamountsofeachcomponentperreaction.Makeenoughmastermixfor(n+1)reactions.

• 2.5l10Xamplificationbuffer
• 2.5l2mMdNTPs
• 1.5l25mMMgCl₂
• 0.12l5U/lTaqpolymerase(0.6Units/reaction)
• 1.25lprimermix(20pMol/lconc.ofprimercombination)
• 14.63ldH₂O

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