Lab 5 Procedure

Prepare Yeast Culture

• Working in a group of 4, obtain ~6 mL culture of yeast strain BY4741 in mid-log phase. You will use the yeast cell for the genome assembly reaction.
• Place 3 microfuge tubes on a rack. Add 1.5 mL of yeast culture to each tube.
• To prepare for the genome assembly reaction, we will first spin down the yeast cells,  “wash” them to remove the culturing media.
• Centrifuge the three tubes at a desktop microfuge at 3000g at room temperature (RT) for 5 minutes. Make sure the centrifuge is balanced before centrifuging.
• Gently remove as much liquid as possible using the 1 mL pipette. Be careful not to disturb the cell pellet.
• Resuspend the pellet in 1 mL of sterile ddH2O. You can resuspend the pellet by pipetting it up and down after adding the ddH2O.
• Centrifuge at 3000g at RT for 5 minutes.
• Repeat two more times and remove as much water from the tubes after the last time of wash.
• Add 100 μL of ddH2O in ONE of the tubes, and resuspend the pellet.
• Transfer the ddH2O + pellet solution to the second tube and resuspend the pellet.
• Repeat the step and resuspend the pellet in the third tube using the same solution. You should end up with a >100 μL of the yeast/H2O solution in the third microfuge tube.
• Keep the tube with yeast cells on a rack while preparing the rest of the reaction.

Assemble the YAC genome assembly reaction

• Take out a new microfuge tube.
• Add 240 μL of 50% PEG 3350 (be slow as this solution is viscous), 36 μL of 1M Lithium Acetate and 50 μL of the Salmon sperm carrier DNA into the new tube.
• Add 34 μL of the PCR product mixture that you prepared from the last lab into the tube.
• Add the content of the tube to the tube containing the yeast pellet.
• Mix well by vertexing vigorously for a few seconds.
• Incubate the tube in a 42 °C heat block for 40 minutes for the YAC reaction to occur.

Plating Yeast Cells

• After 40 minutes, take the tube out of the heat block.
• We will plate the yeast cells in two different concentrations on two plates.
• This quick video explains how to plate bacterial cells (yeast cells are plated the same way):

• For 1 plate, add 200 μL of the reaction on the plate.
• For plate 2, take out a new microfuge tube and add 90 μL of ddH2O into it. Add 10 μL of the YAC reaction into the tube with the ddH2O and add all content of the tube on to the plate.
• Spread both plates using sterile, slightly cooled spreaders. Make sure to use one spreader per plate and sterilizing the spreader between plates.
• Be sure you label your plates with the reaction number, your name and your section. It is best to label the plates around the edge so the writing does not obscure the colonies on the plate.
• Place the two plates upside down in the 30 °C incubator for one or two days.
• The teaming team will take out and store the plates until the next lab period.