Strain Integration

I. Prep work(3 days before starting the protocol, ie “day -3”)

1. Place request for plates
   1. Request “big” agarose plates for mutagenesis (no bacteria on plates, request them for day -1 so that you can use them on day 0)
   2. Request 350 F1 plates for day 3 (need to seed with bacteria, so request them for day 1, and seed so that they are ready to be used by day 3 )
   3. Request 800 F2 plates for day 8 (need to seed with bacteria, so request them for day 6, and seed so that they are ready to be used by day 8)
2. Seed 5 plates, 9 worms/plate of L4’s with array of interest

II. Mutagenesis

1. Chemical
   1. Take Psoralen out of -80 and thaw at RT (WARNING: Psoralen is highly toxic. Wear gloves and keep all psoralen waste in marked containers)
   2. Pick one of the five plates and resuspend all worms in 750 microliters of M9. Collect in eppendorf tube and centrifuge to concentrate worms. Repeat for the other four plates, concentrating ALL worms in a single eppendorf and centrifuging/discarding the excess M9 as need be.
   3. Add Psoralen to a final concentration of 30 ug/ml. Make sure to mix well, as psoralen does not readily go into solution! Our stock is 3 mg/mL, so add 10 ul of Psorelen into 1 mL M9+ worms.
   4. Psoralen is light sensitive, so cover eppendorf with foil and let it sit at RT for 15 minutes.
2. UV
3. 1. Resuspend the worms and pipet into the “big” agarose plate. Move plate around to spread worms.
   2. Make sure the bulbs are on “dark light” (UV bulbs)
   3. Take the plate to UV stratalinker.
   4. Test the UV stratalinker by punching 300 in the microjoules screen (300 x100) and pressing start. Make sure it is counting down correctly
   5. Put in plate and mutagenise at 300 x100 microjoules (Make sure plate is without lid!)
   6. Add one drop of bacteria (to avoid inducing dauer program)
   7. Cover the plate in foil and let it sit at RT for 6 hours
   8. Before picking worms, open the lid and let the M9/Psoralen dry out
   9. Pick 8 L4 worms with the array into each plate (with bacteria) for a total of 10 plates (ie, 80 worms total). Make sure you pick only L4’s: Fewer L4 worms is better than picking young adults. Leave at RT for 3-4 days

III. Integrated line selection

1. On day 1 seed the 350 plates to be used two days from now
2. On day 3 or 4 post-mutagenesis (depending on how worms look) pick 350 F1s and put 1 worm/plate in the plates seeded two days ago. The worms should preferably be should be L4s or early adults.
3. On day 6 seed the 800 plates to be used two days from now
4. On day 7 or 8 post-mutagenesis pick 3 worms from each plate and put each one into its own new, single plate. Make sure to keep the plates ordered in a way that would let you know where each triplet came from (I don’t mark my plates because it is too much work, but keep them in stacks of 9, with 3 sets of 3 plates, that ay I know which ones came from the same parent). Pick a total of 800 plates from the 350 plates seeded before (many of the 350 plates won’t have progeny because the worms will be sick, so end up 3 worms from a total of 250-275 plates).
5. On day 14-15 post mutagenesis I select the homozygote lines by picking plates where all worms have the marker. Note: unc-122:RFP coinjection marker is very variable, so I select all lines where all the adult have the array as homozygotes, no matter how the array looks or if the offspring have the array or not (sometimes it comes early and sometimes late). I have found this is the only sure way to corroborate homozygozity of the unc-122 marker. I don’t throw away slow growing plates: continue to screen until you go through all plates, even the slow growing ones.
6. Freeze integrated lines, backcross them into N2 and map integration