HU recovery assay

HU recovery assay

• Grow 5 ml of cells overnight in YPA + 2% raffinose.
• Dilute overnight cultures to OD (optical density) 0.05 in 4 ml YPA + 2% Glucose (YPAD) or YPA + 2% Galactose (YPAGal) and grow for 1 hour.
• Add 1 ml of 1 M HU (hydroxyurea) in YPAD or YPAGal to the culture. Take 10 µl of the culture for t=0. Grow the cells for 8 h and take 10 µl of culture.
• Dilute the 10 µl from each culture in 990 µl H₂O. Subsequently add 100 µl of this dilution to 900 µl of H₂O and plate out 100 µl of this next dilution in triplicate on YPAD or YPAGal. 
• Colonies were counted by hand after 3 days of incubation at 30°C.
• Viability was calculated by dividing the average amount of colonies (of three plates) after 8 h by the average amount of colonies after 0 h.
• The percentage of survival is normalized to the cell viability of wildtype (glucose or galactose) at timepoint 0. Three individual experiments were performed.

YPA

                                                                                              1 liter                    2 liters  

Bacto peptone (Difco)                                                            20 g                     40 g                       

Yeast extract (Difco)                                                              10 g                      20 g                       

Adenine                                                                                 25 mg                   50 mg   

-              Weigh peptone, yeast extract and adenine

-              Add H₂O to 1 - 2 liter

-              Mix for 10 minutes with a magnetic stirrer 

-              Autoclave for 15 minutes at 115°C

For plates: add 10 grams of agar (Difco) to 500 ml of medium, autoclave as mentioned above, mix and cool to ~65°C, and pour plates (1 liter is ca. 45 plates). Keep the plates for 1-2 days at room temperature, then store them in plastic bags at 4°C in the cold-room.