Preparation of ENU for mutagenesis of C. elegans

Prepared by Jessica Kerins from original by Steve L’hernault

* All steps done in a fume hood

1. Add 100 mL of 10mM acetic acid to 1g ENU, making an 85mM stock solution.

- acetic acid solution made from glacial acetic acid that was filter sterilized

- removed rubber septum of ENU bottle to add acetic acid and a magnetic stir bar

2. Stirred on a magnetic stir plate to dissolve ENU

-.covered bottle with foil

- stirred all day to fully dissolve, checking periodically

3. Aliquot ENU solution into appropriate tubes

- I aliquotted into 16 15mL Falcon tubes, plus 80 0.5mL screw cap tubes: 40 tubes of 100uL, 40 tubes of 200uL.

- all aliquots stored at -20C

- use 15mL aliquots to make additional smaller aliquots, thawing each tube only once; smaller aliquots are also thawed only once

- freezer box of small tubes kept in sealed plastic

- 15mL tube caps wrapped in parafilm, tubes wrapped in foil, kept in plastic bottle

- aliquots at -20C have been used for up to 5 years

4. All waste inactivated in 100mM NaOH for at least 24 hours

ENU Mutagenesis of C. elegans

1. Wash worms as done with EMS mutagenesis, resuspending worm in 3mLs of M9 buffer.

2. From 85mM stock of ENU, prepare 1mL of working stock of ENU and M9 buffer to give desired final concentration of ENU when added to the 3mL worm solution

Ex: For a final concentration of 1.00mM ENU in 4mLs of M9 and worms, make a 1mL working stock of 47.1uL of 85mM ENU and 952.9uL of M9 buffer

3. Add the 1mL of ENU working stock to the 3mLs of M9 and worms.

4. Cover each tube with parafilm and incubate on rotating shaker set at 100 rpm for 4 hours at 20C.

5. Continue as with EMS mutagenesis, inactivating all ENU waste in 100mM NaOH.

* I tried ENU concentrations ranging from 0.3mM to 2.0mM and found that 0.5mM gave the most reasonable mutation rate with the least toxicity.