C. elegans lysis PCR for single worm genotyping.

<aside> ⏱️

Lysis reactions for PCR input are most reliable when used immediately after lysing in the thermocycler. Plan accordingly to avoid prolonged incubation in the thermocycler, or storage on ice or at 4°C, as this may inhibit PCR.

</aside>

<aside> ⚠️

Renumbered during migration. The source had two steps numbered 5, so the protocol ran to 11 with 12 actual steps.

Image path corrected — the picking image was linked as ../img/…, one directory level too high, so it did not display.

</aside>

Materials

• 2X lysis buffer
• 20 mg/mL Proteinase K
• PCR strip tubes and caps
• Thermocycler
• Minicentrifuge
• GoTaq DNA Polymerase (Promega #M3005)
• 5X GoTaq Buffer, green or colorless
• dNTP mix (10 mM)
• Primer pair for gene target (10 μM)
• Nuclease-free water

Protocol

1. Prepare lysis solution by combining 98 μL of 2X lysis buffer with 2 μL of 20 mg/mL Proteinase K. Prepare enough for the number of lyses you want to carry out, at 6 μL per reaction.

2. Aliquot 6 μL of the lysis solution into each cap of an 8-tube strip cap.

3. Pick the desired number of worms into each cap — 3–5 worms per cap is recommended.

   <aside> 🦠

   Take care not to transfer too much bacteria to the lysis buffer when removing worms from the pick. Bacteria will inhibit the downstream PCR reaction. You can watch the pick in the cap through the microscope to make sure the worms come off and to monitor bacterial transfer.

   </aside>

   

   Picking worms into the cap

4. Place the caps onto an appropriately labeled strip tube. Spin the worms down into the strip tube using the minicentrifuge. Double check that the worms reached the bottom of the tubes and are not stuck on the tube walls or in the caps.

5. Replace the flat 8-tube strip caps with domed caps. This helps maintain proper temperature control in the SimpliAmp thermal cycler.