Slot blot to measure DNA repair capacity

To measure DNA damage repair capacity, the genetic material of a bleach-synchronized L1 population is extracted right after damage induction and at a later time point, and immunolabelled for the accumulation of helix-distorting lesions (Mueller et al., 2014). For this experiment, large amounts of synchronized L1 animals (≥ 30.000 per plate) are plated on unseeded 60 mm NGM plates. After UVB treatment at 60 mJ/cm² (broadband), worms are collected in M9 buffer and split into two samples: Sample 1 correspond to the time point 0, in which induced damage remains unrepaired, and Sample 2 is collected 24 hours after irradiation, allowing ample time for DNA repair. Animals of Sample 1 are immediately processed by pelleting in a centrifuge (2 minutes 800 g), the supernatant M9 is removed, subsequently quick-frozen on dry ice or liquid nitrogen and stored at -80°C. Animals of Sample 2 are plated on 60 mm NGM plates seeded with OP50 and incubated at 20°C for 24 hours. After completion of the incubation time, worms are collected in M9, washed 5 times (centrifuge at 200 g for 2 minutes, remove supernatant M9 and add 5 mL of fresh M9) and left 2 hours at 20°C in a rotating mixer to permit the removal of the intestinal bacteria. Thereafter, washing is repeated another 5 times before the sample is pelleted, quick-frozen and stored at -80°C.

For DNA extraction from the samples we can use the Gentra® Puregene® Tissue Kit (Qiagen, 158667) and the protocol for DNA Purification from Tissue. The protocol should be followed as if processing 5-10 mg of tissue (Gentra® Puregene® Handbook, page 39-40). The Cell Lysis Solution is directly added to the thawed sample and the additional step with proteinase K is performed to obtain a maximum yield. The DNA concentration is measured by using the Qubit® dsDNA HS Assay Kit (Invitrogen, {"type":"entrez-protein","attrs":{"text":"Q32851","term_id":"75280859","term_text":"Q32851"}}Q32851). An amount of 2 and 4 µg of DNA can be used to label CPDs or 6-4PPs, respectively. To aid the visualization of the damage, different amounts of DNA are prepared by diluting the DNA serially from six to eight times (1:1). These serial dilutions of DNA are denatured at 95°C for 5 minutes, put directly on ice or at 4°C, and transferred onto a Hybond nylon membrane (Amersham, RPN119B) by using a Convertible Filtration Manifold System (Life Technologies, 11055). To crosslink the DNA, the membrane is incubated at 80°C for 2 hours and then blocked for 30 minutes in 3% milk/PBS-T (0.1%) at RT. To label the lesions, the membrane can be incubated overnight at 4°C with antibodies anti-CPDs (Clone TDM-2, 1:10.000, Cosmo Bio, CAC-NM-DND-001) or anti-6-4PPs (Clone 64M-2, 1:3.000, Cosmo Bio, CAC-NM-DND-002). Posteriorly, the membrane is washed three times with PBS-T (5 minutes, RT) and blocked for 30 minutes with 3% milk/PBS-T. Secondary antibody incubation is performed using a peroxidase-conjugated AffiniPure secondary antibody (1:10.000, Jackson Immuno Research, 115-035-174), followed by 3 washes in PBS-T and incubation of the membrane with ECL Prime (Amersham, RPN2232). Finally, the DNA lesions can be visualized in a Hyperfilm ECL (Amersham, 28906836).