1. Prepare males of your transgenic (30°C 5h, small plates, 5 hermaphrodites, 5 plates, avoid crowding). Adult males will be ready in 3-4 days.

2. Cross the transgenic males with your mutant hermaphrodites (5 males to 1 late L4 hermaphrodite, up to 2 or three hermaphrodites in one small plate) for 2 days.

3. Pick 2-5 hermaphrodites that are positive for your transgene (I assume you can easily observe the transgene) and let them self-fertilize and lay eggs. Eliminate them as soon as you have some eggs to avoid crowding.

4. You should have a couple of homozygous for either one or both transgene and mutation.

5. Proceed to single 20 transgene positive worms, wait for them to have some eggs and genotype the original hermaphrodite for the mutation (primers and reaction conditions are, obviously, up to you and your lab). Keep in mind your primer design needs to differentiate heterozygous from homozygous (e.g. 3 sets of primers or whatever it suits your mutation better)

6. The progeny of the worms that are homozygous for the mutation are going to be either hets or homos for GFP. Just single 2-5 worms from every plate positive for the mutation and observe if their progeny is all positive for the transgene (therefore homozygous).

;)

Try reading http://www.wormbook.org. They have a TON of great resources, including how to set up crosses, screen progeny, etc.

For single-worm PCR, you'll need to pick individual worms into "worm lysis buffer" and extract DNA. I used to use strips of 0.2ml microtubes (same types of tubes as for PCR) and double-checked that there was exactly 1 worm per tube using a dissecting microscope.

Good luck!