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F2 Mutant Screen - non "clonal"

F2 Mutant Screen - non "clonal"

by Michael Koelle, adapted from Erik Jorgenson


1. Mutagenize mixed population worms as per normal. Best to do this first thing in the morning in order to get several lays the next day.

2. Put the whole mixture on plates and let recover 10 minutes (Erik waits 2 hours).

3. Pick L4 animals, put many per plate on new plates. Age the animals 24 hours, at which point they will be early gravid adults. (Note: by aging 24 hours, you avoid the first eggs that the mutagenized animals lay. These may have already been through meiosis at the time of mutagenesis, so that mutations may not have been fixed on both strands of the DNA. By not taking the first progeny, you avoid this theoretical concern. There is also a concern about taking the very last progeny, from third day adults. Here, you may get jackpots, as the mutagenized cells may have undergone several rounds of mitosis before meiosis).

4. Put 3-4 gravid adult P0 per plate on 20 9 cm plates.

5. Move the P0 to new plates every 3-4 hours; this should give you ~50-80 F1 eggs per plate. Do this all day, giving 60-80 total plates. Note: 50-80 F1 on a plate will allow you to get first day adult F2s on the plates before the plates starve out. To get 2nd day adults, need to get less than ~50 F1 on a plate.

An alternative I prefer is to not use serial lays; lots of animals are EMS treated, aged exactly 24 hours after the end of the EMS treatment, and a single lay is taken. This seems to be more convenient. Numbers: Using 3 F1 per plate and a 3 hour lay, the first lay (starting 24 hours after the end of the EMS treatment) will give ~35 F1 per plate. Using 4 F1 per plate, 4 hour lay, starting 24 hours post EMS, get 60-80, average 70, F1 per plate. This will give gravid first day F2 adults with F3 eggs, and then starve soon thereafter, assuming you start with a thick large lawn.

6. Erik's way: Put one batch of 20 plates at 25°. Put the rest at 15° to delay their development, giving you breathing room for the scoring. Move the next set up to 25° after 1.5 days, and the third set to 25° one day later.

6.5 Michael's way: Put one batch at 20°, and two other batches at 15°. After two days, move a 15° batch to 20°. After another two days, move the last batch to 20°. This will space the batches out so that they will be scored 3 days in a row. The last plate will be scored on the 8th day after the EMS treatment.

7. Monitor each set of plates. When the very first F2 L1's begin to hatch, count the F1's on some or all the plates (depending on how uniform the plates are) to estimate the number of genomes screened. (Beth Sawin waits until there are 3-fold embryos and maybe L1's have barely hatched). Each F1 represents 2 haploid mutagenized genomes screened. Then remove the F1 from the plates while leaving the F2 eggs on the plate to develop and later be scored. This is accomplished by dumping some M9 or S basal gently on each plate (use a generous amount that almost fills the plate to the top), swirl gently for 1-2 seconds, and pour off. (If there is a patch of contamination on the plate, cut it off first so as not to spread it). Do this to each of the plates, and set them aside right side up. There will be a few F1 adults remaining on the plate, which will crawl back on the lawn becoming easily visible after a couple of minutes. To remove these, hook up a pasteur pipette to a vacuum line and shove a sterile yellow tip on the end. Under a dissecting scope, using the 12X objective, scan for and suck off the remaining worms. Be careful not to damage the agar suface here or the worms may burrow into the plate.

8. Score the animals at the appropriate time. If later adults are to be scored (e.g. for the Egl phenotype), it may be necessary to modify this protocol for shorter lays in order to keep the plates from going starved.

9. Expect about 4-5 twitchers (unc-22 mutants) per plate if things are going well. Will get a few siblings of each phenotype per plate. You can only keep one animal per plate if you want to know that your mutants are independent. However, pick several animals per plate, since about 1/3 of the animals will be sterile perhaps due to other mutations. It is Erik's impression that the very first lay doesn't give as many mutants as the next couple; perhaps the very first germ cells to mature aren't EMS mutagenized as well as earlier stage germ cells.