EMS mutagenesis

Prepared by Jessica Kerins from unknown original

 

*When working with EMS (or ENU), always work in fume hood and wear lab coat, goggles, and gloves.

 

 

1.  When plates have at least 50% L4s, wash 6 plates with M9 buffer (1 mL/plate) and transfer with a glass pasteur pipette to two 15mL conical tubes.

 

2.  Fill tubes with M9 buffer and spin in clinical centrifuge (setting 4, or 1800 rpm) for 4 minutes

 

3.  Aspirate supernatant and fill each tube with 6 mL M9 buffer.  Spin again.

 

4.  Aspirate supernatant and fill each tube with 4 mL M9 buffer.

 

5.  In fume hood, pour 20-25 mL of 4N NaOH in a 50 mL conical tube, for disposal of EMS-contaminated tips.

 

6.  In fume hood, with a filtered tip, add volume (see table below) of EMS stock (stored at RT in fume hood) to the 15 mL conical tube of worms + M9.  Shake thoroughly.  Decontaminate tip by disposing in the tube of NaOH. 

 

Final EMS concentration

Volume of EMS stock added to tube

25 mM

10uL

37.5 mM

15uL

50 mM

20uL

62.5 mM

25uL

 

We generally use a final EMS concentration of 50 mM or 62.5 mM and find that these concentrations give a reasonable mutation rate and little toxicity. 

 

7.  Incubate tubes for 4 hours at 20¡C on a rotating shaker set at 100 rpm.

 

8.  Spin tubes as above.  Remove supernatant with pasteur pipette, leaving 1 mL in the tube, and dispose both supernatant and pipette in NaOH.

 

9.  Add 6 mL of M9 buffer to tubes and spin as above.  Remove supernatant, leaving 0.5 mL in the tube, and decontaminate both supernatant and pipette as above. 

 

10.  Transfer remaining 0.5 mL onto a NGM plate (1 plate/tube).  Let dry with lid ajar for 30 minutes in fume hood, or until all EMS has evaporated.  Decontaminate conical with NaOH. 

 

11.  Transfer three L4s/plate onto 30 fresh plates and incubate at 20¡.  Let self, then pick one L4/plate (as many plates as possible - we usually do about 3 big boxes of plates Å 400-500 plates) and incubate at your desired screening temperature.  Screen the next generation for an F2 screen.

 

12.  Dispose NaOH/EMS solution down the fume hood sink.  Dry waste can be disposed of normally once all EMS has evaporated.