UW Stout/D2O SP22

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Revision as of 11:04, 26 April 2022 by Teagueb (talk | contribs) (Pilot Experiment with Wild Type Yeast Cells)
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Introduction

D2O also known as Heavy Water is a form of water that is deuterium. It is heavier than normal H2O and can have many physical and chemical properties that are different than normal H2O. It is known to slow down neutrons and, in some cases, possibly absorb these neutrons. Although it is similar to water it can only be somewhat safely consumed in very small quantities. The experimentation process uses a Wild-Type yeast cell as a control and 9 Knockout Strains for the experimentation. With this, we will observe and record the growth curves of these cells using Optical Density 600 in a 24-hour observation period which in return will tell us the number of cells in each well plate.

Materials and Amounts

  • 1ml D2O
  • Sterile Water
  • Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
  • Corning COSTAR 96-well clear flat-bottom assay plate
  • 9 Knockout Yeast Strains

Equipment

  • Biosafety Cabinet
  • Micro-pipetting tips
  • Micropipettes
  • Molecular Devices Spectra Max Plus 384 Microplate Reader

Safety

  • Safety Glasses
  • Lab Jacket
  • Gloves
  • Safety Data Sheet for D2O

Procedure

  1. Diluting the D2O with sterile water, by following the dilution rates below
    1. 50% dilutions (25ul D2O to 25ul H2O) (10 samples of this dilution)(Used only in Pilot)
    2. 0% dilutions (50ul H2O) (10 samples of this dilution)(Control)
    3. 75% dilutions (37.5ul D2O with 12.5 ul of H2O)(Used only in Pilot)
    4. 35% Dilutions (17.5ul D2O with 32.5ul of H2O)(Final Dilution)
    5. 20% dilutions (10ul of D2O with 40ul of H2O)(Used only in Pilot)
  1. Vortex the yeast culture briefly to resuspend the yeast cells.
  2. Set up the plates as 50ul of dilution a with 50ul of Yeast cells (Well 1-9) and dilution as 50ul dilution and 50ul H2O (Well 10)
  3. Set up the plate reader as follows:
    1. Temperature: 30°C
    2. Mode: Kinetic
    3. Wavelength: 600 nm
    4. Interval: 5 minutes
    5. Total run time: 24 hours
    6. Shake before reading: 30 seconds
  4. Transfer the assay plate to the reader and read for 24 hours.

Pilot Experiment with Wild Type Yeast Cells

  • On 4/12/2022 we did a pilot experimentation process with a 50% dilution and control. They showed that the OD 600, had good results (inserted below). They showed an increase in the OD 600 and a turn in the number of cells in the well of the plate compared to the control dilution. This is telling us that cells are growing less in the D2O diluted with water rather than in the pure water control (B3).B3- Control A3-50% dilution

4.14.2022.png

  • We ran a pilot experiment with a control of 75%, 35%, and 20%. This was to fill in the gray area of our last pilot experiment and this led us to see some great information and data to help us with our overall experiment. A3 was 75% dilution. B3 was 35% dilution, C3 was 20% dilutions, and D3 was the control at 0% dilution. This showed again an increase in the OD 600 and that the number of cells in the well decreased. It shows that as the dilution increased so did the OD 600 of each dilution and it perfectly fills in the areas of the previous graph. As you can see the OD 600 decreases and then seems to plateau during the 24 hours. With the higher, concentration of the dilution came to a lower OD 600 value.

4.19.2022.png