Yeast Genetics Complementation Experiment: Procedural Flow Chart
1. Record the color of the parental strains of yeasts.
Accumulation of certain products in the enzyme pathway making adenine result in different pigmentations. Recording the color of the different strains of yeasts at first will help us later analyze the complementation results.
2. Prepare YPD plates of the parental strains for mating. Using two YPD plates – one for A and one for Alpha – stripe 3 different lines with the three different strains on each plate. Make sure to sterilize the scraper on a Bunsen Burner every time!
In this step, we are preparing our parental colonies to grow so that next week, we can cross them and see if the colonies are still able to grow and therefore deduce complementation patterns between the different strains of A and Alpha yeasts. Sterilizing the scraper is important for preventing cross-contamination of the yeasts. We want to make sure we are looking at each strain separately, so the Bunsen Burner should be used to completely sterilize the scraper being used to transfer the yeast colonies onto the YPD plates.
3. Place YPD plates in 30oC incubator.
The plates should incubate for about a week in order for the colonies to grow to a good experimental size.
4. Mate the A and Alpha plates by replica plating the stripes perpendicular to each other on a YPD plate. (Exact steps are outlined in following procedure.)
This is where the complementation tests begin. By replica plating, we can transfer the specific yeasts to another spot where we want them and then mate them to see if complementation occurs between different strands.
5. Label a fresh YPD plate with arrows indicating North and East, for aligning type A and type Alpha respectively.
The arrows will be used to align the lines on the two YPD plates containing the parent strains. This will orient us in the right direction when replica plating, keeping us from getting confused when performing the mating between the different strands.
6. Using sterile velvet squares, transfer the lines of yeast from one plate onto the YPD plate being used for complementation. Press the velvet cloth lightly on the bottom of the dish in order to transfer the yeast to the velvet. Use the arrows to orient yourself when pressing the velvet back down onto the mating plate.
This will allow us to mate the different strains. The velvet should not...