EMS Mutagenesis Protocol
Ann Moon
DAY1:
1. Grow liquid culture (> o/n) at 26˚C.
2. Take 1.0ml of culture, spin and wash 2x in 5ml sterile 0.1M NaPi, pH6.6.
3. Resuspend in 1.5ml 0.1M NaPi.
4. To each of 3 tubes add 0.2ml of cell suspension. Add 1.0ml of NaPi.
5. To tubes 2 and 3 add 50µl EMS (Ethyl Methanesulfonate, an alkylating agent). Final conc =
4.2%.
6. Shake 1 & 3 for 70 min. at 30˚C, and 2 for 50 min. at 30˚C.
7. Transfer 0.4ml of each treated (or untreated) cell suspension to 8.0ml of 6% Na-thiosulfate.
8. Let cells sit 10min.
9. For each suspension, dilute in sterile ddH2O 10-4 (0.1 into 10ml, 0.1ml into 10ml).
10. Spread 0.1, 0.2, 0.4ml of each on YPD plates.
11. Collect remaining cells and freeze in YPD with 25% glycerol (-70˚C), 1ml total.
12. Allow to grow for 5 days and analyze. An example is given below.
DAY6:
# Viable and % Kill
0.4ml
0.2ml
0.1ml
#
%
#
%
#
%
1. control
-
-
158
-
29
-
2. 50 min. EMS
-
-
12
92
7
76
3. 70 min. EMS
7
-
4
97
2
93
avg=84%
Work with EMS2 -- want to plate 200/plate on YPD on 5 plates........calculations:
Concentrated 8 fold before freezing
Got 6.3 colonies/100µl at 10-4 dilution of original
Want 200/plate, would use 3.2ml of original 10-4 dilution
Now is 8x concentrated, would use 0.4ml of 10-4 dilution
Will use 0.1ml of 4 x 10-4 dilution (1/2500)
Dig into bottom of frozen stock (assume homogeneous -- don't want to rethaw, bad for mutants).
Dilute 0.05ml into 5ml, 0.2ml into 5ml
Control plate: 0.1 into 10, 0.1 into 10, 1 into 8.
DAY16:
Analysis of DBY877 Mutagenesis Efficiency:
Non-mutagenized controls: even colony size, some petites.
(on 5 plates)
RED COLONIES:
0 /473
(0%)
PETITES:
35/473
(≈7%)
Mutagenized: huge colony size disparity.
RED COLONIES:
4/835
(≈0.5%)
since there are 2 genes that give ade-, get
a mutgenesis frequency of 0.25%.
Assuming ≈2000 genes in yeast, each
cell should have at least 5 mutations.
PETITES:
Assume at least 8% of mutants are petites. Many small colonies
may not have time to express ade- phenotype, so mutation
frequency may even be higher. In fact, on DAY17, 3 more reds
are found. NOTE: Of all red mutants, 3 are sectoring.
SAFETY NOTE: EMS is highly carcinogenic. It is stored at 4˚C. Always work in the hood with
gloves. When incubating at 30˚C, it is important to put the tubes on a roller because EMS is
"oily" and does not mix well with water. EMS is volitale, so use tightly capped tubes (such as
orange cap 15ml dispo screw capped tubes), and also parafilm over the cap. Na thiosulfate
inactivates EMS (but EH & S still want it disposed through them).
Helpful Reference: Lindegren, Hwang, Oshima, Lindegren (1965). "Genetical Mutants Induced by
Ethyl Methanesulfonate in Saccharomyces," Can. J. Genet. Cytol. 7:491-499.
5/6/93 A. Moon
based on procedure by D. Drubin (1/22/86), obtained via D. Vinh