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        MMS integration

        互联网

        769

        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 MMS integration l in 1 ml) MMS in M9.
        2. Put 100 MMS integration 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 MMS integration 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 MMS integration 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

         

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