From : Anita Sil; Janet Chenevert
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.
5ml overnight in YEPD.
OD600 of overnight sample. Ideally want to mutagenize 200,000,000
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.
cells and wash with 1.5 ml 0.01M KPO4, pH 7.0.
cells and resuspend in 1.5 ml same buffer.
cells for 5 seconds.
sample into one 1 ml aliquot and two 0.25 ml aliquots in eppendorf tubes.
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
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.
cells. Transfer supernatant to EMS waste. Wash twice with
1 ml water. Wash once with 1 ml YEPD.
in 1 ml YEPD. Remove 10 ul to plate 10-3, 10-4, 10-5 dilutions to
judge kill curve.
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.