High Efficiency Yeast Transformation
High efficiency yeast trafo with LiAc
Start a preculture in a big flask containing 5mL of YPD the previous day. The next day Add 100 to 200 mL of YPD per 5mL of preculture and wait about 5-6 hours (your culture need to have about 2 to 3 doubling time in fresh media). You can check OD after 1h and at the end. Depending of the strain optimal OD600 might be between 0.3-0.6
- Usually, I use 300mL (6x 45mL) of cells for 10 trafos.
- Harvest 45mL of cells in a 50mL falcon tube by centrifugation 5-7 min 4500g, RT..
- During the centrifugation you can denature ssDNA for 20 min at 96°C and chilled 5min in ice.
- Discard all YPD and resuspend cells in 45 ml water, and spin down cells for 5-7 min at 4500g ,RT
- Discard all the liquid (pour and pipette off the rest). Combine 2 pellets of cells together and resuspend cells in 40ml water, add 5mL 10x LiAC-TE buffer, mix by inverting the tube. (optional, incubate cell in LiAc for 20 min) and spin down cells for 5-7 min at 4500g ,RT
- Discard most the liquid by pouring off only. And combine all cells one tube, you need 2.1mL of cells, if not enough, add some 1x LiAc to get 2.1mL.
- Use 2mL tube for transformation Per tube : (final concentration : 34%PEG, 1xLiAc-TE) Add 700µL of PEG 50%, 82µL 10x LiAC-TE and mix with a 1mL pipette or votex. 10µL of DNA Add 208µL cells and 30µL denatured ssDNA(8-10µg/µL) and mix immediately with the 1mL pipette (Everything is done at RT except for the ssdna which should be maintained in ice until adding to the cells)
- Optional if you want you can do a master mix with PEG, LiAc-TE, Cells and ssDNA and aliquot by 1.020 mL PEG/LiAc-TE/Cells/ssDNA in 2mL eppis containing 10µL of dna of interest.
- Incubate the mix for 30 min at 28°C
- Heat shock at 42°C for 15 min (time can be slightly different, up to 20-30 min, depending of the yeast strain)
- Incubate cells again at 28°C for 30-60 min (if you need to incubate your cells in YPD, go to next step now)
- Pellet cells at 5000g for 2min
- Pipette out PEG solution and resuspend cells in 1mL of water (or TE pH7.5) to wash out PEG.
- Pellet cells at 5000g for 2min
- Resuspend cells in your favorite final volume (water or TE). And plate your cells. In case you want to incubate your cells in YPD, resupend them in 1mL YPD and incubate them for 1-2h at 28°C. Pellet cells at 5000g for 2min
- Pipette out YPD solution and resuspend cell in 1mL of water (or TE pH7.5) to wash out YPD.
- Pellet cells at 5000g for 2min
- Resuspend cells in your favorite final volume (water or TE). And plate your cells.
- PEG50% : 50% W/V PEG 4000 from Serva
- LiAc-TE : 1M LiAc, 20mM NaEDTA, 100mM Tris-HCl/Tris-Base, pH 7.5 pH is ajusted by mixing both Tris (no NaOH or HCl), ie: for 100mL : dissolve LiAc, and NaEDTA in 90mL H20 and adjust pH to 7.5 by filling up to 100mL with 1M Tris-HCl and 1M Tris-Base solutions.
D. Loque March 2007
Yeast Cell Perfusion using Cellasic Platform
Cellasic device to Study trapped yeast cells perfused with different solutions
1. Prepare the plate. Make sure you use the appropiate pore size for your cells (S, M, L) adding the buffer in the first two wells (A) F1 adding the 4 different solutions to perfuse (different concentration of glucose) F2 to F5 adding the cells cultures at a concentration of 1-20 106 cells/ ml (this ranges around 0.065 to 1.3 OD)
2. Connections: Plug and start laptop where the Cellasic software is installed (black laptop in the lab), start cellasic software. Get the cellasic hardware (blue box), plug and prepare all the conexions before turn on
a-USB conection to the laptop
b-Vacuum system: connect the white-end pipe to the blue box where it says vacuum, turn on the pump in the white box
c-Air pump: connect other white-end pipe to the blue box where it says air, then connect that wider pipe to the thinner pipe that can be connected to the multi division air system. In the multidivision air system, make sure you open the valve of the exit you chose (black small valve). Connect the multidivision air input pipe to the pipe out of the room that is connected to the air bottle (the left one). Open the air bottle (using grey wheel and the black bottom valve) to reach 20 psi in the meter (help your self in the regulation with the other two black regulators). Once you have air going in the main pipe, you can regulate the air pressure that will go in the sytem using a black regulator (in top of the multi division system). When you move this up you can select your highest pressure in the system (entering the blue box). You can use a maximum of 10 psi. Make sure the green thin pipe that connects to the multidivision system is well connected, it tends to move and loose the air in there.
3-Turn on the blue box.
4-Sealing the plate: The plates are 96-well shaped and once one is loaded with the solutions it should be vacuum-sealed to the adaptor (manifold) that is connected to the blue box. Once they are attached click in the button that says vacuum (in the manual option) in the control area (right side) in the cellasic software. Push down on the manifold with slightly force for 5 sec to ensure uniform contact during sealing. You will notice the plate and the adaptor are now sealed. Now its necessary to place this in the microscope using an appropiate adaptor (black says Frommer lab). There is an specific one for the 96 well-plates. To place it in the microscope you need to slide it towards you and make sure it doesn’t move when you touch it. It should be very fixed (there is a fixation point in the downer left corner). Once this is fixed, the plate with the vacuum adapter can be placed there.
5-Turn on all the microscope and imaging devices: the big three boxes behind the table in the left self over the screen the box Lambda 10-2 on the microscope table black box Quantem the brown box on the table that is attached to the microscope that says quantem ensure the blue wide part connecting microscope to the brown box has the black top down. To turn on white light in the microscope is in the shutter button in the front of the microscope
6-Use tape and plastilin to adjust the plate to the black platform so the cells in the images taken don’t move when pumping different solutions.
7-Focus on the center of the imaging area where the cells should be trapped. Use the 40x objetive with inmersion oil. With this objetive only one of the two cell trapping regions can be seen. Using the 20x could allow to analyze both areas at the same time (check this)
8-In the manual options from the cellasic software let some flux go through so all the piping in the plate is filled with buffer first. Let it flow for a while (recommended 5 min at 6psi to flush out shipping solution). After washing turn off both flow switches. To do so go in the manual section of cellasic software and fill the desired pressure for the wells selected and then click in the well you want to start pumping (the grey color will change)
9-Turn on ‘cell load’ to transport cells into the trapping area. Suggested loading protocol: 6-10 PSI for 10 seconds. But this depends on the cell density and cell profile (especially the size) and desired trapping density. Click in the cells well so the grey color changes
10-Turn on flow with the buffer solution to remove the cells that are not trapped. After 5 minutes of flow , the remaining cells will be those firmly held in for imaging.
11- Using the automatic flow control section to generate a program with the steps of each of the solutions. Most likely is a combination of lower concentration of glucose followed by buffer and then higher concentration, buffer and so on.
Before you activate the program to start, the imaging and photography software should be prepared using the slidebook
Yeast RNA extraction
- Make up β-ME/RLT (10ul β-ME per 1ml RLT). 600ul per sample
- Vortex pellet in 600ul β-ME/RLT .
- Add this to ~600ul acid washed beads in a 2ml tube.
- Shake on tissuelyzer (2min, 1 minute at a time with a 1 minute chilling interval)
- Extract sample from tube by inserting needle into bottom of tube and spinning down into fresh tube.
- Spin down this supernatant for 2 min at max speed.
- Load this onto gDNA eliminator column. Spin 30s @ >10Krpm.
- Add 1 volume 70% EtOH, mix with pipettor.
- Load onto column, 700ul at a time, spin @ >10krpm. Discard flow thru.
- Add 700ul RW1, spin @ >10Krpm 30s. Discard the flow thru.
- Add 500ul RPE, spin @ >10Krpm 30s. Discard flow thru.
- Add 500ul RPE, spin @ >10Krpm for 2 min. Discard flow thru.
- Place column in a 2ml collection tube and spin at max speed for 1 min.
- Place column in a 1.5ml collection tube.
- Add 30 to 50ul RNase-free water, spin at >10Krpm for 1 min to elute. (Reload elutate and spin again to improve yield.)
revised by Frommer Lab 2012
V. Spotting Assay to Detect Effector-induced Growth Inhibition Phenotypes
- From a fresh plate pick a yeast colony (1-2 mm diameter) carrying the expression plasmid of interest and inoculate, in a 15 ml polypropylene tube, 3 ml of synthetic complete medium supplemented with 2% glucose as a carbon source and without the amino acid or nucleotide to which the expression plasmid provides prototrophy. Repeat the same procedure for a control yeast strain carrying an empty plasmid. Place the culture tubes in a roller and incubate overnight at 30°C with constant rotation.
- In the following day, remove the cultures from the roller and centrifuge at 800 g for 5 min at room temperature. Remove the tubes from the centrifuge and discard the supernatant.
- Resuspend the cell pellet in 3 ml of sterile DDW and mix by vortexing.
- Repeat the centrifugation step and resuspend cells in 3 ml of sterile DDW.
- To determine their optical density (OD), transfer 100 μl of each culture to a microtube containing 900 μl DDW and vortex. Pour the content of the tubes into a plastic cuvette and measure the absorbance at a wavelength of 600 nm (use as reference a cuvette filled with 1 ml of DDW). Calculate the OD600 of the initial cultures by multiplying the OD600 of the cultures in the cuvette by a dilution factor of 10.
- Next, based on the OD600 of the initial cultures, prepare cell suspensions (1 ml) with an OD600=1.0 in sterile microtubes.
- By using 3 sterile microtubes filled with 900 μl sterile DDW, prepare three 10-fold serial dilutions (OD600=0.1, 0.01 and 0.001) from each cell suspension (OD600=1.0) prepared in the previous step.
- Prepare two plates containing synthetic complete medium without the amino acid or nucleotide to which the plasmid of choice provides prototrophy. The medium of one plate should be supplemented with 2% glucose and that of the other plate with 2% galactose and 1% raffinose. Dry the plates in a sterile laminar flow hood at room temperature for 20 min and place them on a grid.
- For each culture, spot 10 μl from the four dilutions in a row on both the repressing and inducing media.
- After spotting, leave the plates open in the hood for several minutes. When the spots are dry, cover the plates and place them in a 30°C incubator for 2-3 days.
- After incubation, take out the plates and analyze yeast growth. First, confirm that the cell densities are similar on the plate containing repressing medium. Then, compare the growth of the culture expressing the effector protein of interest with the culture carrying an empty vector on the plate containing inducing medium.
revised by Frommer Lab 2012