DUAL LUCIFERASE ASSAY
Overnight Phase:
1) Retrieve 4 overnight tubes per strain (shelves right of microwave), 5mL disposable pipets (black rack left of monitor row), and Pipet Aide (throughout workstations).
2) Label 4 tubes per strain (MM####, genotype, mating type, initials, date). Number tubes for each strain 1-4.
3) Clean benchtop with 70% ethanol and light Bunsen Burner with flint.
4) Pipet 3.333 milliliters C-Ura into each tube numbered #1, 3 milliliters C-Ura into each tube numbered #2 and #3, and 2.7 milliliters C-Ura into each tube numbered #4.
5) Pull needed strains from -80 Degree Celsius freezer (see protocol on Wiki).
6) Prepare an overnight tube (see protocol on Wiki, YPD is substituted with C-Ura) in tube #1 for each strain.
7) Return yeast strains to freezer.
8) Make Serial Dilutions (for a ratio of 1000:100:10:1):
- Flame rim of tube #1
- Pipet 333 microliters yeast suspension from tube #1 into tube #2. Flame rim and vortex gently.
- Pipet 333 microliters yeast suspension from tube #2 into tube #3. Flame rim and vortex gently.
- Transfer 333 microliters yeast suspension from tube #3 into a flip-top tube.
- Pipet 300 microliters yeast suspension from flip-top tube into tube #4. Flame rim and vortex gently.
- Repeat for each strain.
- Dispose of flip-top tubes in tip waste.
9) Place tubes in 30 Degree Celsius shaker at an angle. Shake overnight.
10) Turn off flame and clean benchtop with 70% ethanol.
Minimal Media Preparation Phase:
1) Clean benchtop by magnetic stirrers with 70% ethanol.
2) Retrieve following materials (ALL MUST BE STERILE IE. MARKED WITH BLACKENED AUTOCLAVE TAPE):
[1] 2 Liter Beaker (bottom left glassware shelf) [1] 250 milliliter or 500 milliliter Graduated Cylinder (second or top right glassware shelf) [1] Stir Bar [2] Spatulas [2] Weigh Boats Electronic Scale
3) Label 2L beaker with tape and Sharpie (Min, Initials, Date).
4) Place 2L beaker on magnetic stirrer and insert stir bar.
5) Measure 250 milliliters milliQ water at sink. Pour into beaker. Replace foil cover and set stirrer to low speed.
6) Zero electronic scale with weigh boat and measure 1.7 grams Yeast Nitrogen Base (stored alphabetically).
7) Transfer Yeast Nitrogen Base to 2L beaker, scraping with spatula and tapping gently as necessary. Replace foil cover on beaker.
8) Zero electronic scale with new weigh boat and measure 5 grams Ammonium Sulfate (stored alphabetically).
9) Transfer Ammonium Sulfate to 2L beaker. Replace foil cover on beaker.
10) Measure and transfer 750 milliliters milliQ water to beaker and set stirrer to moderate speed. Allow all solutes to dissolve completely.
11) Transfer volumes of 250 milliliters into [4] 500 milliliter glass bottles (bottom or second left glassware shelf). Label bottles (Min, Initials, Date)
12) Prepare bottles for autoclave by loosely capping and marking with green-and-white-striped autoclave tape (on autoclave cart)
13) Dispose of weigh boats in trash can, rinse spatulas and beaker, and clean benchtop with 70% ethanol
14) Autoclave (protocol found on Wiki)
Amino Acid Solution Preparation:
1) Prepare to make Amino Acid Solutions by gathering following materials:
[5] EMPTY 50 milliliter conical tubes (green caps and stored above workstations) [5] 50 milliliter conical tubes of Distilled Water (top solutions shelf) [5] STERILE Spatulas (Drawer 59) [5] Small Weigh Boats (Drawer 61) Electronic SmartWeigh Scale (Drawer 61) Tube Racks (Cupboard 66)
2) Label empty conical tubes on side (Valine 100X, Initials, Date; Isoleucine 100X, Initials, Date; Lysine 100X, Initials, Date; Histidine 100X, Initials, Date; Leucine 50X, Initials, Date) and top (Valine 100X, Isoleucine 100X, Lysine 100X, Histidine 100X, Leucine 50X).
3) Zero scale with small weigh boat, and measure .2929 grams Valine (top shelf above magnetic stirrers)
4) From one tube of dH2O, pour approximately 15 milliliters dH2O into appropriately labeled empty conical tube. Add Valine carefully and add remaining water to 50 milliliter line. Close tube securely with screw cap and shake to dissolve.
5) Zero scale with small weigh boat, and measure .328 grams Isoleucine (top shelf above magnetic stirrers)
6) From one tube of dH2O, pour approximately 15 milliliters dH2O into appropriately labeled empty conical tube. Add Isoleucine carefully and add remaining water to 50 milliliter line. Close tube securely with screw cap and shake to dissolve.
7) Zero scale with small weigh boat, and measure 1.4619 grams Lysine (top shelf above magnetic stirrers)
8) From one tube of dH2O, pour approximately 15 milliliters dH2O into appropriately labeled empty conical tube. Add Lysine carefully and add remaining water to 50 milliliter line. Close tube securely with screw cap and shake to dissolve.
9) Zero scale with small weigh boat, and measure 1.556 grams Histidine (top shelf above magnetic stirrers)
10) From one tube of dH2O, pour approximately 15 milliliters dH2O into appropriately labeled empty conical tube. Add Histidine carefully and add remaining water to 50 milliliter line. Close tube securely with screw cap and shake to dissolve.
11) Zero scale with small weigh boat, and measure .656 grams Leucine (top shelf above magnetic stirrers)
12) From one tube of dH2O, pour approximately 15 milliliters dH2O into appropriately labeled empty conical tube. Add Leucine carefully and add remaining water to 50 milliliter line. Close tube securely with screw cap and shake to dissolve.
13) Dispose of empty conical tubes in orange Biohazard waste bucket and weigh boats in trash can, rinse spatulas, and clean benchtop with 70% ethanol.
Drug Stock Phase:
1) Clean benchtop by magnetic stirrers with 70% ethanol.
2) Gather following materials:
[1] 15 milliliter conical tube (blue top, upper shelves throughout workstations) [1] Small Weigh Boat (Drawer 61) [1] Sterile Spatula (Drawer 59) Electronic SmartWeigh Scale (Drawer 61) [1] 10 milliliter Disposable Pipet (black rack left of monitor row) Pipet Aide (throughout workstations) Tube Rack (Cupboard 66)
3) Zero SmartWeigh scale with small weigh boat and mass out .05 grams 8-aza-A (glass container in 4 Degree Celsius refrigerator).
4) Pipet 3 milliliters DMSO in 10mL disposable pipet and dispense into 15mL conical tube. Replace sterile wrap on pipet when finished.
5) Carefully transfer 8-aza-A into 15mL conical tube.
6) Pipet and dispense 7 milliliters DMSO into 15mL conical tube with 10mL disposable pipet.
7) Securely cap 15mL conical tube with blue screw-top. Shake gently to dissolve drug.
8) Dispose of pipet in biohazard waste bucket and clean benchtop with 70% ethanol. Keep drug refrigerated if it will not be used immediately (4 Degree Celsius refrigerator).
Optical Density Phase:
1) The next morning, retrieve serial dilutions from shaker.
2) Gather following materials before moving to Hayek/Parra Lab (other side of room)
P-1000 Micropipets P-1000 Micropipet Tips
3) Start machine? UV 2.5 Program…Wavelength (WL) set at 600 nanometers
4) Clean benchtop with 70% ethanol
5) Pipet 1 milliliter C-Ura into a cuvette to “blank” the machine
6) Orient cuvette such that machine's light shines through clear plastic (DO NOT TOUCH CUVETTE BELOW RIM). “Blank” machine with Autozero function.
7) Pipet 1mL each sample into cuvette (one at a time, new tips and cuvettes each time) and record absorbence. Return each sample to original tube when finished. Machine is only accurate below a reading of 1.0, so samples with higher readings must be calculated from a dilution as explained below:
- Pipet 500 microliters sample into cuvette
- Pipet 500 microliters C-Ura into cuvette
- Measure optical density of diluted sample
- Multiply absorbence reading by two and record as original, undiluted sample's absorbance
8) Clean benchtop and remove materials from Hayek/Parra Lab.
Resuspension Phase:
1) Calculate what volume of the most appropriate sample would be needed to create a 4mL suspension with absorbence of .8
Ex. Sample #2 = .441
.441(Volume)=.8
Volume=.8/.441
Volume=1.81mL
2) Clean benchtop with 70% ethanol and light Bunsen Burner.
3) Pipet calculated volume of each sample into its own 15mL conical tube. Add enough water to make the volumes of each conical tube equal.
4) Spin down using large centrifuge in Hayek/Parra Lab.
5) Carefully remove supernatant (cell pellet will be very small) from each conical tube.
6) For the Wild Type 8-aza-A Control, resuspend pellet in 3.96 milliliters MinD+ media and add 40 microliters 8-aza-A drug solution.
7) For all other samples, resuspend in 4 milliliters MinD+ media.
8) Transfer each sample suspension into its own glass culture tube and incubate in shaker for 4 hours (set timer for 3 hours to allow time for more preparatory steps).
9) Turn off flame and clean benchtop with 70% ethanol.
Buffer Preparation Phase:
Buffer Kit is stored in -20 Degree Celsius Freezer, but since the Kit is expensive, Christine will prepare the buffers after 3 hour timer has sounded. Set a 1 hour timer at that point.
Second Optical Density Phase:
Repeat Optical Density Phase steps, this time calculating for an OD600 of .22/mL as shown below:
Sample #2 =.33/mL
.33(Volume)=.22
Volume=.22/.33
Volume=.667mL
Second Resuspension Phase: