Jan 02, 2025

Public workspaceGenotyping C. elegans

DOI
dx.doi.org/10.17504/protocols.io.yxmvmebbbg3p/v1
  • 1Arcadia Science
  • Arcadia Science
Arcadia Science
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DOI: dx.doi.org/10.17504/protocols.io.yxmvmebbbg3p/v1
Protocol Citation: Justin Donnelly 2025. Genotyping C. elegans. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmebbbg3p/v1
Manuscript citation:
Avasthi P, Borges AL, Cheveralls K, Donnelly J, Mets DG, Reiter T. (2025). An experimental and computational workflow to characterize nematode motility behavior. https://doi.org/10.57844/arcadia-b89a-7e76
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 23, 2024
Last Modified: March 13, 2025
Protocol Integer ID: 106384
Abstract
This protocol describes a procedure to genotype C. elegans strains via genomic extraction and PCR amplification. You should always confirm strain information upon receipt of the strain.
Materials
ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S ReagentMagnesium ChlorideFisher ScientificCatalog #AC223210010 ReagentPotassium chlorideP212121 ReagentTris HClP212121
PCR tubes
Thermocycler
Platinum wire
PCR master mix
PCR primers
1% agarose DNA gels
ReagentNew 6x Purple Loading DyeNew England BiolabsCatalog #B7024S
Ultrapure water
Protocol materials
ReagentNew 6x Purple Loading DyeNew England BiolabsCatalog #B7024S
ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
ReagentMagnesium ChlorideFisher ScientificCatalog #AC223210010
ReagentPotassium chlorideP212121
ReagentTris HClP212121
ReagentNew 6x Purple Loading DyeNew England BiolabsCatalog #B7024S
ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
ReagentTris HClP212121
ReagentPotassium chlorideP212121
ReagentMagnesium ChlorideFisher ScientificCatalog #AC223210010
Genomic extraction from single worms
Genomic extraction from single worms
15s
15s
Prepare lysis buffer. You can prepare lysis buffer in advance and store it at TemperatureRoom temperature before adding proteinase K.

Lysis buffer (without proteinase K):
Concentration10 millimolar (mM) ReagentTris HClP212121
Concentration50 millimolar (mM) ReagentPotassium chlorideP212121
Concentration2 millimolar (mM) ReagentMagnesium ChlorideFisher ScientificCatalog #AC223210010
Ph8.3

Complete lysis buffer by adding ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S to a final concentration of Concentration0.5 mg/mL . Prepare ≥Amount10 µL complete lysis buffer per sample.

Transfer Amount10 µL lysis buffer per tube to PCR tubes. Include an additional tube as a negative control.
Pick a single worm from each strain and transfer to lysis buffer
Flame-sterilize a platinum wire. Pick up one adult worm from plate, taking care to prevent puncturing the NGM. Transfer worm by gently submerging the platinum wire into the PCR tube under flame. Hold the wire in this position for several seconds to allow worms to detach. Flame-sterilize the wire before picking additional worms.
Note
Try to pick only a single worm per PCR tube. A couple of worms is fine, but too many can inhibit the reaction.

Immediately spin tubes in a benchtop microcentrifuge for Duration00:00:15 .

15s
Freeze tubes in LN2 or Temperature-80 °C for ≥Duration00:10:00 .

10m
Lyse worms in a thermocycler according to the following protocol:
Temperature65 °C , Duration01:00:00 –Duration01:30:00 .
Temperature95 °C , Duration00:15:00 (proteinase K inactivation step).

You can store extracted worm DNA indefinitely at Temperature-80 °C .

2h 45m
PCR amplification
PCR amplification
If using poison primer amplification, add Amount46 µL Q5 PCR master mix per tube to appropriate number of PCR tubes. To each, add Amount2 µL template (proteinase K extraction product) and Amount0.5 µL each primer.

If using standard amplification, add Amount47 µL Q5 PCR master mix per tube to appropriate number of PCR tubes. To each, add Amount2 µL template (proteinase K extraction product) and Amount0.5 µL each primer.

For negative control, add master mix and primers only.

Run PCR for 30–35 cycles according to Q5 manufacturer protocol. Determine annealing temperature based on primer sequences.
Analysis of PCR product via gel electrophoresis
Analysis of PCR product via gel electrophoresis
Transfer Amount20 µL each PCR product to a new PCR tube and add Amount4 µL ReagentNew 6x Purple Loading DyeNew England BiolabsCatalog #B7024S .

Load onto 1% agarose DNA gel. Electrophorese at Amount120 V until dye front reaches the end of the gel.

Visualize under blue light.
Note
For large-scale deletion strains with validated poison primer sequences, gel analysis is usually sufficient to confirm genotype. In other cases, further analysis via Sanger sequencing is required.

Analysis of PCR product via sequencing
Analysis of PCR product via sequencing
In a clean PCR tube, add Amount9 µL ultrapure water, Amount1 µL PCR product, and Amount1 µL sequencing primer.

Submit sample for Sanger sequencing.
Protocol references