SingleWormPCR

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Reagents:

LysisBuffer:	50mMKCl 10mMTrispH8.3 2.5mMMgCl2 0.45%NP-40(IGEPAL) 0.45%Tween-20 0.01%Gelatin
ProteinaseK:	20mg/ml
10XPCRBuffer:	100mMTris,500mMKCl,15mMMgCl2pH8.3
dNTPmix:	25mM/each
primers:	5-10uM
TaqPolymerase:	approx5U/ul

Procedure:

1. AddproteinaseKtolysisbuffer(95ullysisbuffer+5ul20mg/mlproteinaseK)
2. Place3uloflysisbufferintopof200ulPCRtube
3. Picksinglewormintolysisbuffer
4. Spindowntobottomoftubebyspinninginmicrofuge15seconds@14,000rpm
5. Freezetube@-80atleast1hour
6. LysisofwormandreleaseofgenomicDNA
   • heattubeto65degreesfor60-90minutes
   • inactivateproteinaseKbyheatingto95degreesfor15minutes
7. PerformPCR
   • add27ulofPCR"mastermix"toeachtube
   • mastermix:1XPCRBuffer,0.5uMprimers,0.2mM/eachdNTPs,Taqpolymerase
   • runPCRreactionfor30-35cycles

References:

• AgeneticmappingsysteminCaenorhaBDitiselegansbasedonpolymorphicsequence-taggedsites.WilliamsBD;SchrankB;HuynhC;ShownkeenR;WaterstonRH.Genetics,1992Jul,131(3):609-24.
• Cloning,sequencing,andmappingofanalpha-actiningenefromthenematodeCaenorhabditiselegans.BarsteadRJ;KleimanL;WaterstonRH.CellMotilityandtheCytoskeleton,1991,20(1):69-78.

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