Single Worm PCR

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Reagents:

Lysis Buffer:	50 mM KCl 10 mM Tris pH 8.3 2.5 mM MgCl2 0.45% NP-40 (IGEPAL) 0.45% Tween-20 0.01% Gelatin
Proteinase K:	20 mg/ml
10X PCR Buffer:	100 mM Tris, 500 mM KCl, 15 mM MgCl2 pH 8.3
dNTP mix:	25 mM/each
primers:	5-10 uM
Taq Polymerase:	approx 5U/ul

Procedure:

1. Add proteinase K to lysis buffer (95 ul lysis buffer + 5 ul 20mg/ml proteinase K)
2. Place 3 ul of lysis buffer in top of 200 ul PCR tube
3. Pick single worm into lysis buffer
4. Spin down to bottom of tube by spinning in microfuge 15 seconds @ 14,000 rpm
5. Freeze tube @ -80 at least 1 hour
6. Lysis of worm and release of genomic DNA
   • heat tube to 65 degrees for 60-90 minutes
   • inactivate proteinase K by heating to 95 degrees for 15 minutes
7. Perform PCR
   • add 27 ul of PCR "master mix" to each tube
   • master mix: 1X PCR Buffer, 0.5 uM primers, 0.2 mM/each dNTPs, Taq polymerase
   • run PCR reaction for 30-35 cycles

References:

• A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites. Williams BD; Schrank B; Huynh C; Shownkeen R; Waterston RH. Genetics, 1992 Jul, 131(3):609-24.
• Cloning, sequencing, and mapping of an alpha-actinin gene from the nematode Caenorhabditis elegans. Barstead RJ; Kleiman L; Waterston RH. Cell Motility and the Cytoskeleton, 1991, 20(1):69-78.

compiled by Chad Rappleye

Aroian Lab Protocols