UW Stout/Formamide FA19
Haley and Thomas
Formamide Sensitivity Torture Experiment
Written by Haley Monette and Thomas Lo
Reasoning: To observe the growth of the yeast cells by adding different concentrations of formamide.
Background: Since previous experiments succeeded with the concentration of the formamide being 3%, we decided to test a range surrounding that mid-point.
We will test .5 increments above and below 3%, ranging from .5%-5.5%, measuring 12 concentrations.
Neg Control- No formamide meaning 50 ul yeast and 50 ul water.
- 600ml of concentrated yeast cell solution (OD 600 0.2)
- Assay plate (has 96 wells)
- “Neat” (full conc.) formamide 33 ul
- PCR Tube
- 567ul of H2O
- Fume Hood
- Incubator set to 30 degrees Celsius
Excessive inhalation of vapor may cause symptoms that parallel ingestion, ranging from headache to unconsciousness, depending upon the duration and level of the exposure. May cause headache, dizziness, nausea, vomiting, abdominal pain, and unconsciousness.
- Make sure the H2O is sterile.
- Make sure everything you use in the experiment is sterile, including yourself (spray everything down with 70% ethanol).
- Formamide is best accessible when taking 40 ul and putting it in a separate tube, apart from the bottle.
- Make sure to use a different tip each time you grab water, yeast, or formamide.
- Return bottle of formamide to frig when done.
- Grab the bottle of neat formamide and pipet out 36 ul into a PCR Tube.
- Obtain 561 ul of H2O in a beaker.
- In the fume hood, dilute out each yeast solution with desired formamide concentration. *Refer to chart below (Desired formamide concentration mixtures)*
- Take each concentration (50ul) and combine that with 50ul of yeast concentration into a single well of the assay plate.
- Negative control well (#12)- 50ul yeast and 50 ul water.
Note: Only 12 wells will be used, each different concentration of the formamide will have its own well.
- Incubate assay tray for 12-24 hours at 30 degrees Celsius.
- Record growth in each well.
Desired Formamide Concentrations:
- 0.5 ul formamide + 49.5 ul H2O
- 1.0 ul formamide + 49 ul H2O
- 1.5 ul formamide + 48.5 ul H2O
- 2.0 ul formamide + 48 ul H2O
- 2.5 ul formamide + 47.5 ul H2O
- 3.0 ul formamide + 47 ul H2O
- 3.5 ul formamide + 46.5 ul H2O
- 4.0 ul formamide + 46 ul H2O
- 4.5 ul formamide + 45.5 ul H2O
- 5.0 ul formamide + 45 ul H2O
- 5.5 ul formamide + 44.5 ul H2O
- 50.0 ul H2O + 50.0 ul Yeast Concentration (Negative Control Well)
We used well C on the assay plate.
By using the doubling-time we can tell how fast the yeast strains are growing,
t= start and end times
q= growth in OD600 at those times
Wells 4-11 did show any growth.
Our graph has shown that our concentration of formamide at 1% (well #2) showed the best promising growth. This is the concentration we will stick with and apply it to our chosen knock out strands.
Procedure for each K.O Strand:
Add 1.0 ul formamide + 49.0 ul H20 into 50.0 ul of Yeast Concentration for wells 1-11 on the assay plate and incubate at 30 degrees Celsius for 12-24 hours.