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, with gloves and lab coat.
- Add 100 mL of 10mM acetic acid to 1g ENU, making an 85 mM 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
- Stirred on a magnetic stir plate to dissolve ENU
-.covered bottle with foil
– stirred all day to fully dissolve, checking periodically
- Aliquot ENU solution into appropriate tubes
– I aliquotted into 16 15 mL Falcon tubes, plus 80 0.5 mL 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
- All waste inactivated in 100mM NaOH for at least 24 hours
ENU Mutagenesis of C. elegans
- Wash worms as done with EMS mutagenesis, resuspending worm in 3mLs of M9 buffer.
- 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 uL of 85 mM ENU and 953 uL of M9 buffer
- Add the 1mL of ENU working stock to the 3 mLs of M9 and worms.
- Cover each tube with parafilm and incubate on rotating shaker set at 100 rpm for 4 hours at 20C.
- 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.