UW-Stout/Hydroxyurea FA19

Hydroxyurea Dilution and Concentration Protocol

Materials and Equipment

• Yeast
• Powdered Hydroxyurea
• Sterile Water
• Micropipette
• 1.7 ml microcentrifuge tubes
• 96 Well assay plate
• Analytical Balance
• Vortex Mixer

Protocol

1. The solution will have to be made fresh every day because the hydroxyurea will start to decompose in an aqueous solution. Hydroxyurea is also only soluble up to 50mg/µl.
2. To create your stock solution of 50mg/µl hydroxyurea, you will need to take the powdered hydroxyurea and mix it with 1.14 ml of water.
3. Take your hydroxyurea and dilute it to create 5mg/µl and 0.5 mg/µl of stock solution using the following equation
   1. (Final Concentration/Stock Concentration)*Final volume=Volume of Stock
4. Each well will be filled up with 50 microliters of yeast, and 50 microliters of hydroxyurea solution. The well on the farthest left will contain the highest concentration of hydroxyurea at 50 mg/µl
5. Add 50mg/µl of yeast into each well
6. Dilute out your hydroxyurea for each well. There are 12 wells and they should each contain the following concentrations from left to right

Yeast(µl)

50 50 50 50 50 50 50 50 50 50 50 50

Hydroxyurea(mmol)

40 30 20 10 40 30 20 10 40 30 20 10

Stock Solution(mg/µl)

50 50 50 50 5 5 5 5 0.5 0.5 0.5 0.5

Water in µl

10 20 30 40 10 20 30 40 10 20 30 40