C. elegans Single Worm PCR¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
Single Worm PCR for use with C. elegans
Lysis Buffer* 1 X PCR buffer (see below for 10X PCR buffer) 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
- Add proteinase K to 1 X PCR buffer (95 ul 1 XPCR buffer + 5 ul 20mg/ml proteinase K)
*We do not use the “Lysis buffer” recipe anymore because 1 X PCR buffer seems to work fine. *
- Place 5-10 ul of 1 X PCR buffer + proteniase K in top of 200 ul PCR tube (if you use more than 1 worm use 10ul instead)
- Pick single worm (or multiple worms) into lysis buffer.
- Immediately (don’t let sit too long in lysis buffer) spin down to bottom of tube by spinning in microfuge 15 seconds @ 14,000 rpm or just flick down.
- Freeze tube in Liquid Nitrogen at least 10min.
- Lysis of worm and release of genomic DNA (You can use the PCR machine –”worm lysis” program)
- Heat tube to 65 degrees for 60-90 minutes
- Inactivate proteinase K by heating to 95 degrees for 15 minutes. If you do not use the worm DNA right away store at -80C.
- Perform PCR (50ul reaction)
- Add 45-40 ul l of PCR “master mix” to each tube
- Master mix: 1X PCR Buffer, 0.5 uM primers, 0.2 mM/each dNTPs
- Add 0.5ul of Taq to tubes at 95C (hot start) If you have many samples the master mix can include the Taq so you can skip this step.
- Run PCR reaction for 30-35 cycles
Williams BD, Schrank B, Huynh C, Shownkeen R, Waterston RH A genetic mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged sites. Genetics (1992) pmid:1321065