1. Pick 100 L4 worms containing the array of interest into an empty plate. If you have several arrays of the same construct, use one which has a low transmission rate, as it will require less generations to isolate an integrant.
2. Prepare 30ug/ml TMP in M9. Our stock is 100X. Note: TMP should be remade fresh every 6-12 months (dilute the powder in DMSO and heat to dissolve). Spread 1-2 ml on the plate with the worms until they are soaked in it. Do this in the fume hood. Don’t overfill to avoid spills – TMP is very toxic. Cover with the lid and with foil (TMP is light sensitive) and wait 15 minutes.
3. Irradiate the worms with 300uJoules in the Stratalinker in the Luo lab. Make sure to use dark light (365 nm) bulbs and to remove the lid from the plate. Use “energy” rather than “time” settings to achieve 300 uJoules.
4. Put the plate back in the hood with the cover removed so the TMP would evaporate. Add a drop of bacteria so the animals don’t starve. Wait 1-2 hours for evaporation.
5. Pick the worms into four or five regular OP50 plates.
6. Let the worms grow until they finish the food (~2 days on the bench).
7. Using a sterilized scoop, concentrate as many worms as possible in the middle of the plate, then chunk that area to a new plate.
8. Perform several rounds of steps 6 and 7.
9. Examine the plate under the fluorescent scope. When the frequency of the marker has dropped sufficiently, single 20 worms per plate and check in the next generation if they are integrants (I.e. 100% of progeny should contain the coinjection marker).
The number of cycles you’ll need to do will depend on how fast your array is lost, that’s why using lower transmission arrays is better. The original protocol says to wait until 1/500 worms shows the co-injection marker. We have found that you can get an integrant well before that stage, so use your own judgment to decide when to start looking for the integrants on a plate. The C.Kenyon protocol says it can 4-15 generations.In any case, don’t throw away the plate when you single worms from it – keep it at 16C should you want to go back to it to “dilute” the array further.
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