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MMS integration
MMS integration
(both methods modified by Herman Lab)

NOTES:

  • Remember that MMS is a potent mutagen -- wear gloves and be very careful.
  • Integration is best done using an array that is transmitted at very low frequency.
  • MMS has a short aqeuous half-life

Henderson Method -- approx. 1-5% integrants

  1. Make a solution of 0.1% (1 l in 1 ml) MMS in M9.
  2. Put 100 l of 0.1% MMS in a thumb depression plate.
  3. Pick 35-40 L4 non-Uncs into the 0.1% MMS.
  4. Incubate for 10 min at room temperature (RT).
  5. Transfer mutagenized worms to individual plates (TIP).
  6. Rinse all items with LOTS of water.
  7. Pick 100-120 F1 L4/YA non-Uncs TIP.
  8. Pick 6 L4/YA non-Uncs TIP from 100 F1 plates (total 600 F2 plates). Don't try to score plate for % transmission.
  9. Screen F2 plates for 95% non-Uncs.
  10. Pick 6 L4/YA non-Uncs TIP from F2 candidate plates.
  11. Screen F3 plates for 100% non-Uncs. The stack of six plates should all be close to 100% at this point.
  12. Pick 6 L4/YA non-Uncs TIP from F4 candidate plates.
  13. All six F4 plates should be 100% non-Uncs.
  14. Start backcrossing.
Koelle Method -- approx. 1-3% integrants
  1. Make a solution of 0.1% (1 l in 1 ml) MMS in M9.
  2. Transfer 150 L4 non-Uncs to a plate.
  3. Wash worms off using M9 into a 1.7 ml bullet.
  4. Spin in mini centrifuge 20-30 sec.
  5. Remove supernatant.
  6. Add 100 l of 0.1% MMS solution.
  7. Incubate for 10 min at room temperature (RT).
  8. Transfer mutagenized worms to individual plates (TIP).
  9. Rinse all items with LOTS of water.
  10. Pick 520 F1 L4/YA non-Uncs TIP over two days.
  11. Pick 2 F2 L4/YA non-Uncs TIP from each F1 plate. Need to pick 1030-1050 plates since 3-5 % of worms are dead or sterile. Don't try to score plates for % transmission but if one looks close to 100% pick 4 F2 TIP. Don't label plates; keep each generation in labeled boxes.
  12. Screen F2 plates for 95% non-Uncs over two to three days (18-20 hours).
  13. Pick 4 L4/YA non-Uncs TIP from F2 candidate plates.
  14. Screen F3 plates for 100% non-Uncs. The stack of four plates should all be 100% at this point.
  15. Pick 4 L4/YA non-Uncs TIP from F4 candidate plates.
  16. Confirm 100% non-Uncs and start backcrossing.