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 t o i ndividual p lates (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 t o i ndividual p lates (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