Difference between revisions of "UW-Stout/Hydroxyurea FA19"

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'''Yeast Knockout Strand with Hydroxyurea Protocol'''
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Materials:
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# Yeast Strands
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# Powdered Hydroxyurea
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# Sterile H2O
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# 96 Well Plate
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Procedure:
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# Make 50mg/µl of the stock solution with 0.056g of powdered hydroxyurea and 1.14ml of H2O
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#Then dilute it down to make 300µl of the 5mg/µl solution
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## You will be using this to replicate series 7 from the previous experiment above
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# After you have made your solution put in 50µl of each yeast strand in the wells
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# Next put in 20µl of the hydroxyurea solution and 30µl of the H2O in each well
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#Once all of the wells are filled, put the plate in the SpectaMax Plus 384 Microplate Reader

Latest revision as of 07:46, 12 December 2019

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

wtgraph.png


Yeast Knockout Strand with Hydroxyurea Protocol

Materials:

  1. Yeast Strands
  2. Powdered Hydroxyurea
  3. Sterile H2O
  4. 96 Well Plate

Procedure:

  1. Make 50mg/µl of the stock solution with 0.056g of powdered hydroxyurea and 1.14ml of H2O
  2. Then dilute it down to make 300µl of the 5mg/µl solution
    1. You will be using this to replicate series 7 from the previous experiment above
  3. After you have made your solution put in 50µl of each yeast strand in the wells
  4. Next put in 20µl of the hydroxyurea solution and 30µl of the H2O in each well
  5. Once all of the wells are filled, put the plate in the SpectaMax Plus 384 Microplate Reader