Integration protocol (Modified from Greenwald & Hobert labs):
Stratagene UV Stratalinker 2400 (254 nm)
- irradiation: Set the machine to 300 mjoules (x100).
Irradiate: 100 worms on 20 6 cm plates (normal worm plates), i.e., 5 worms per plate. You can also irradiate a large plate of animals, and pick L4s onto 20 plates after the irradiation.
Herman lab modification:
Pick 100s of L4s onto one plate, expose to UV and allow to recover for 24 hours. Then transfer healthy adults to 20 fresh plates (5 adults per plate) and allow to starve at 20ˇC for 2 weeks, etc.
[Usually we irradiate 20 plates and grow half at 20oC and half at 15oC. We screen the first 10 plates, and if we donŐt get an integrant, then we go to the next 10 plates. However, we generally get integrants from the first 10 plates.]
- starve worms
on the plates: 1-2 weeks for the 20oC worms (2 wks is better
than 1 wk), 1 1/2 weeks for the 15oC worms
- chunk (~1 cm) starved worms on fresh plate. Take one chunk from each plate.
We initially only chunk the 20oC worms; the 15oC worms are chunked only if needed.
- 1 day later: pick 200-300 transgenic animals (the ones showing best expression of the marker) onto individual plates. Generally, we initially pick 10-15 animals from each master plate onto individual plates (for a total of 100-150 plates). If we donŐt get any integrants, then we go to our second set of master plates, the ones that have been kept at 15oC. In the case of GFP transgenics, pick the brightest, non-mosaic animals.
- wait for two generations and then score the plates - on average 3-4 of those picked animals will be homozygous for the integration. You will also have a lot of heterozygous plates from which you can isolate homozygous integrants. For transgenics that have the rol-6 marker, you can score after one generation. However, with the GFP lines, we find that sometimes we get a decrease in the number of fluorescent animals in the F2 generation, so itŐs easier to wait and just screen the F2 animals.
Markers successfully used:
GFP reporter constructs (such as Pflp-4::gfp or Psur-5::GFP)