RobWardenWNB:M3D2

Expression Engineering, Day 2, 4/6/07

Counting Yeast

Purpose

To find the number of yeast in a liquid culture. Later we will compare this to the count of viable yeast that grow on a plate.

Protocol

1. Vortex the culture tubes to fully resuspend the yeast.
2. Label two eppendorf tubes “ON” and "log".
3. Remove 100 ul of each culture to the appropriate eppendorf tube.
4. Add 900 ul of water to each eppendorf tube.
5. Invert the tubes several times to mix the contents. Holding the eppendorf tube upside-down, you should flick the bottom of the tube to mix-in the last drop of liquid.
6. Dilute the cultures to a final dilution of 1:100 by making another 1:10 dilution of your 1:10 dilutions.
7. Pour 1 ml each dilution 1:100 and 800 ul of each 1:10 dilution into a cuvette and fill a fifth cuvette with 1 ml water.
8. Read the optical density of each sample at 600 nm, using the cuvette of water to blank the spectrophotometer.
9. Consider your OD600 values that are within the reliable range of the spectrophotometer (generally considered to be from 0.1 to 1.0). Determine the concentration of your undiluted yeast cultures using the relationship of 1 OD600 = 1 x 10^7 cells/ml. Don’t forget to account for the dilutions you made.

Results

Phase	Dilution	OD600	Count
Overnight	1:10	0.0809	8.09 x 10^6
Overnight	1:100	0.0076
Log	1:10	0.4769	4.769 x 10^7
Log	1:100	0.0472

Plating Yeast

Purpose

To plate yeast so that we can count them later and determine the viability of the yeast

Protocol

1. Shake 8 eppendorf tubes onto the benchtop then close the caps and put them in your rack. Label them ON/10^-1, /10^-2, /10^-3, /10^-4, /10^-5, and log/10^-1, /10^-2, /10^-3.
2. Use your P1000 to add 900 ul of sterile water to each.
3. Vortex your undiluted cultures then use your P200 to remove 100 ul and add it to the appropriate “10^-1” eppendorf. Close the cap and invert to mix, flicking in the last drops when the eppendorf is upside-down.
4. Use the 10^-1 dilutions to make your 10^-2 dilutions and repeat until you have serially diluted to 10^-5 of the original concentration.
5. Plate 100 ul of your ON/10^-5 dilution onto one YPD, one –trp, one –ura.
6. Plate 100 ul of your log/10^-3 dilution onto another YPD, another -trp, another -ura.
7. Wrap your plates with your colored labeling tape and incubate them, media-side up in the 30° incubator, until next time.

Competent Cells

Purpose

To prepare the yeast cells for transformation.

Protocol

1. Add 1ml Sterile Water into an eppendorf tube
2. Scrape FY569 Cells off of plate and add to tube. Vortex to mix the cells evenly.
3. Spin these cells at 3000 rpm for 1 minutes in the microfuge.
4. Remove the supernatant by aspirating. You do not have to remove every drop.
5. Wash the cells with 1 ml "wash solution".
6. Harvest the cells in a microfuge, spinning 1 minute at full speed.
7. Aspirate the supernatant.
8. Resuspend the pellet in 150 ul of "competent solution". Split between 3 tubes.

Yeast Transformation

Purpose

To insert our PCR product into the FY569 genome. This should knock out SPT8 and knock in URA3 so that our yeast are viable on SC-ura media and express the SALSA complex.

Protocol

1. Add 5 ul of "no template" PCR reaction to one eppendorf and label the top appropriately. This should serve as your "no DNA," negative control. Flick the tube to mix the contents.
2. Add 5 ul of pRS416 DNA (50 ng) to another eppendorf and label appropriately.
3. Add 5 ul of your "plus template" PCR product from last time. This is your experimental sample. You can give the remainder of your PCR products to the teaching faculty who may run them on an agarose gel, depending on the outcome of these transformations.
4. To each tube add 500 ul "transformation solution" to your cells. Vortex the tube to make an even suspension.
5. Incubate the tubes at 30° for approximately one hour, along with 4 SC-ura petri dishes, with their lids ajar if there is moisture on their surface. Periodically "flick" your tubes to mix the contents, this will help keep the cells from settling to the bottom.
6. After at least an hour, flick the tubes to mix the contents and then spread 250 ul of each mixture on your SC-ura dishes, plating the experimental transformation twice.
7. Wrap your plates with your colored labeling tape and incubate them, media-side up in the 30° incubator until next time.

Summary

Today we practiced some basic yeast techniques. This will allow us to further study yeast in the future. Specific to our project, we transformed the yeast cells with our PCR product. Colonies we see later on the SC-ura plates will have incorporated our construct into yeast genome, and knocked out the SPT8 gene. Our yeast will then express the SALSA complex, and we can characterize the phenotype associated with this SAGA-like complex.