Experiments

1. Use a permanent marker to label the top of a 2 ml centrifuge tube with your number.
2. Transfer 1 ml of the E. coli overnight culture into the tube.
3. Place it into a balanced centrifuge and spin for 1 min at 12000 rpm, pour off the supernatant. Be careful not to disturb the pellets.
4. Invert your tube and tap gently on the surface of a clean paper towel to drain thoroughly.
5. Add 250 μL of Buffer P1 to your tube. Vortex to resuspend the pellet. Check that no cell clumps should be visible.
6. Add 250 μL of Buffer P2 and mix gently by inverting the tube 6-8 times. Solutions will become relatively clear. Do not allow the lysis reaction to proceed for more than 5 mins.
7. Add 350 μL of Buffer P3 and invert the tube immediately but gently 6-8 times. The solution should become cloudy. Shake the tube gently to disperse the precipitate.
8. Spin for 10 mins at 12000 rpm to pellet the precipitate thoroughly.
9. At the same time, label a spin column on the top and by the side. Balancing the column by applying 500 μL of Buffer BL, spin 1min at 12000 rpm. Discard the flow-through.
10. Apply 600 μL of the supernatant from step 9 to the spin column by pipetting, spin 1min at 12000 rpm. Discard the flow-through.
11. Add 600 μL of Buffer PW to the column, sit for 1min. Spin 1 min at 12000 rpm. Discard the flow-through.
12. Repeat step 11. Discard the flow-through. Put the column back, spin 3mins at 12000 rpm to remove the residual wash buffer.
13. Get a clean 1.5 mL centrifuge tube and label the top. Place the column into this tube, add 50 μL of Buffer EB to the center of the column, let stand at 50℃ for 5 mins. Spin 3 mins at 12000 rpm.
14. The concentration of plasmid is measured by nanodrop.

3.1 PCR Mix Preparation

3.2 Initial Incubation

Place the tube into PCR machine, set 55°C for 1 hr.

3.3 Ligation Program

Add 0.5 μL T4 ligase into the tube. Place the tube into PCR machine and run the following program:

1. Fill the ice box with ice, and put E. coli Competent Cells (50μL per tube) to make it melt on ice.
2. Add 2μL plasmid to one tube of 50μL E. coli Competent Cells. Incubate the tube on ice for at least 30 min.
3. Set the water bath to 42℃ and place the tube with E. coli into the water bath for 30-90 seconds (Heat shock).
4. Immediately place the tube back on ice and incubate for 5 min.
5. In an ultra-clean table, add 250μL pre-warmed LB culture medium to the E. coli centrifuge tube and tap the bottom of the tube to mix well.
6. Incubate at 37℃ for at least 45 min with shaking to allow expression of resistance genes.
7. Using a pipette, take 100μL shaken bacteria from the centrifuge tube and add it to the LB plate marked with Antibiotics. Use sterilized glass beads and apply evenly.
8. Discard the glass beads and incubate those plates upside down overnight at 37℃ in a constant temperature incubator.

5.1 Preparation of Competent Cells

Grow yeast cells (BY4741) at 30°C in 10 ml YPD broth until mid-log phase.

1. Pellet the cells at 500 x g for 4 minutes and discard the supernatant.
2. Add 10 ml Frozen-EZ Yeast Solution 1 to wash the pellet. Repellet the cells and discard the supernatant.
3. Add 1 ml Frozen-EZ Yeast Solution 2 to resuspend the pellet.

5.2 Transformation

1. Mix 50 μl of competent cells with 0.2-1 μg DNA (in less than 5 μl volume); add 500 μl Frozen-EZ Yeast Solution 3 and mix thoroughly.
2. Incubate at 30°C for 45 minutes. Mix vigorously by flicking with finger or vortexing 2-3 times during this incubation.
3. Spread 50-150 μl of the transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading.
4. Incubate the plates at 30°C for 2-4 days to allow for growth of transformants.

6.1 Pick and Incubate Single Colony Bacteria

In the laminar flow hood, add 1 ml of LB liquid medium containing the appropriate antibiotics into a 1.5 ml EP tube, and use the pipette tip to pick (white) single colonies and discard them into the centrifuge tube. (Pick 10-15 single colonies from each plate.)

6.2 Incubation

Incubate at 37°C with 220 rpm shaking for 3 hours on a shaker. Proceed with single colony PCR validation.

6.3 PCR Mix

6.4 PCR Program

Mix the PCR system using a vortex mixer, and centrifuge shortly. Then place the PCR tube in the thermal cycler and run:

1. Measure 1g of agarose, mix it with 100mL of TAE.
2. Microwave for 1-3 minutes until agarose dissolves.
3. Pour the mixture into a gel tray with well comb in place.
4. Let the mixture cool down for 10-15 minutes.
5. Once solidified, place the agarose gel into a gel box.
6. Load samples and marker into the wells of the gel.
7. Put the gel into the electrophoretic chamber.
8. Start the electrophoretic chamber for 20 minutes, 130V.
9. Remove the gel afterwards and place it in a UV light source.
10. Compare the sample markings to the marker.

8.1 Fermentation Conditions

1. 8.1.1 Seed Culture Preparation
   • Inoculate a single colony of S. cerevisiae within YPD medium and cultivate overnight, then the seed solution would be obtained.
2. 8.1.2 OD600 Measurement
   • Measure the OD₆₀₀.
3. 8.1.3 Main Culture
   • The seed culture was inoculated to an initial OD₆₀₀ of 0.1 into a fertilized 250 mL flask containing 50 mL SC-URA medium.
   • Cultivate at 30℃ and 250 rpm.