This report shows a method of using half of a brood of worms as the source of genomic DNA to perform PCR following backcrossing of knockout lines. The figure below shows the breeding strategy used.

5 plates were set up with 3 him-5 males and 1 T27A3.1(tm1572) L4 hermaphrodite ( Approximately 3 days later, the parents were discarded and the plates were observed for males as an indication of cross-progeny. From the original plates, 5 L4 hermaphrodites were picked and placed onto individual plates (step 4). The genomic DNA was phenol-chloroform extracted from the original plates and PCR performed to confirm that heterozygous worms were produced.

The 5 L4 hermaphrodites were moved to new individual plates and allowed to continue their egg laying. The plates containing the first half of the brood were used for DNA extraction and PCR. The second plates for which the first plate PCR confirmed the presence of heterozygotes were kept. These +/- worms self-fertilized to produce 25% homozygous knockout. One additional transfer of worms was needed (step 11) and the worms from step 10 were genotyped. These F2 worms that were T27A3.1 -/- were then individually bred with the wild-type males (him-5) and the process was begun again. This procedure was repeated 3 more times.

The figure below shows an example gel following PCR performed from the phenol-chloroform extracted genomic DNA (A). The bands for the heterozygotes are clear but when single worm PCR was performed (B), the results were inconsistent and the bands were faint. In this circumstance, we used males generated from a cross of 3 males and 1 hermaphrodite so that we knew that all the male offspring resulting should be heterozygotes.