Single Worm PCR

Reagents: 

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

Procedure:

  1. Add proteinase K to 1 X PCR buffer  (95 ul 1 XPCR buffer + 5 ul 20mg/ml proteinase K) *Note: We do not use the "Lysis buffer" recipe anymore because 1 X PCR buffer seems to work fine.
  2. 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)
  3. Pick single worm (or  multiple worms) into lysis buffer.
  4. 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.
  5. Freeze tube in Liquid Nitrogen  at least 10min.
  6. 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.
  7. 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

 

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.

 

modified form  Chad Rappleye

Aroian Lab Protocols