Herskowitz Lab Protocol
From : Anita Sil; Janet Chenevert

EMS Mutagenesis

Solutions

A. 0.01M KPO4 pH 7.0,
0.39 ml 1M monobasic stock,
0.61 ml 1M dibasic stock,
H2O to 100 ml.

B.  5% sodium thiosulfate, filter sterilized, 100 ml, make fresh.

Mutagenesis/Kill Curve:

1. Grow 5ml overnight in YEPD.

2. Take OD600 of overnight sample.  Ideally want to mutagenize 200,000,000 cells/ml.

3. Transfer 1.5 ml cells into an eppendorf tube.  May need a larger volume if OD of cells is low.  I used 1.5 ml of cells around OD600 = 0.4.

4. Pellet cells and wash with 1.5 ml 0.01M KPO4, pH 7.0.

5. Pellet cells and resuspend in 1.5 ml same buffer.

6. Sonicate cells for 5 seconds.

7. Split sample into one 1 ml aliquot and two 0.25 ml aliquots in eppendorf tubes.

8. (For the zero timepoint of mutagenesis, plate 10-3, 10-4, and 10-5 dilutions of one of the 0.25 ml aliquots.)

9. Add 20 ul EMS to the 1ml aliquot of cells.  Place eppendorf tube in top of large glass roller drum tube.  Cap the tube, and then parafilm shut.  Place tube at 30°C.  Also place remaining 0.25 ml aliquot of cells (with no EMS added) at 30°C under the same conditions.

10. Remove 0.25 ml aliquots from the 1ml tube at various timepoints:  30 min, 60min, 90 min, 120 min.  To stop the reaction at each timepoint, add 1ml 5% sodium thiosulfate.  For the final timepoint, also treat the unmutagenized 0.25 ml aliquot in parallel.

At Each Timepoint:

11. Pellet cells.  Transfer supernatant to EMS waste.  Wash twice with 1 ml water.  Wash once with 1 ml YEPD.

12. Resuspend in 1 ml YEPD.  Remove 10 ul to plate 10-3, 10-4, 10-5 dilutions to judge kill curve.

When doing actual mutagenesis, one will pick one or two timepoints based on the kill curve.  Once the cells are finally resuspended in YEPD, the next step will be to plate the cells out so they can be assayed for the screen.  If one is doing a visual screen, the mutagenized cells should be split up into many pools and grown in YEPD for 24-36 hours AFTER mutagenesis to bias against dead or dying cells.