The overall goal of this procedure is to transform yeast cells with an array library of plasma DNAs. This is accomplished by first growing up an appropriate amount of yeast cells. The yeast cells are then treated with lithium acetate to make them competent for DNA transformation.
Next, transform the competent e cells by mixing them with DNA. The final step of the procedure is to select and analyze transformants. Ultimately, results can be obtained that show the effects of the plasma DNA on yeast phenotypes through yeast spotting assays and or microscopy.
The main advantage of this technique over existing protocols, such as standard yeast transformations, is that it allows for the transformation of a large number of different Plasmids into yeast cells in an automated fashion. This high throughput yeast Transformation protocol is designed for 10 96 well plates, but can be scaled up or down accordingly. For larger scale experiments, the key to success is to keep all plates and reagents organized.
Once the experiment is underway to avoid confusion, A biobot rapid plate liquid handler will be used in this protocol To begin aliquot, five to 10 microliters of plasma DNA from an array yeast plasmid library into each well of the round. Bottom 96 well plates. Place the 96 well plates uncovered inside a clean bench fume hood overnight to dry.
Next inoculate 200 milliliters of yeast peptide dextrose or YPD media with a query strain in a 500 milliliter baffled Lin Meyer flask incubate overnight at 30 degrees Celsius with shaking on the following morning. Dilute the query strain to an OD 600 of 0.1 in two liters of YPD media for optimal growth. Grow up to one liter cultures in separate 2.8 liter baffled flasks.
Shake the flasks for four to six hours at 30 degrees Celsius until the cultures reach an OD 600 of 0.8 to 1.0. When the yeast Culture has reached an OD 600 of 0.8 to 1.0, harvest the cells by centrifugation. Fill four disposable 225 milliliter conical tubes with culture, and spin a 3000 RPM for five minutes in a tabletop centrifuge.
Pour off the S supernatant from each tube. Add more culture to the same tubes and centrifuge again, repeating as necessary until all cells are harvested. Wash each cell pellet in 50 milliliters of autoclave distilled water and Resus spend by vortexing.
Combine cells in one two hundred and twenty five milliliter conical tube and spin at 3000 RPM for five minutes. After removing the S supernatant, wash the cells in a hundred milliliters of lithium acetate solution. Spin again at 3000 RPM for five minutes.
Remove the SNA resuspend the cell pellet in 70 milliliters of lithium acetate solution. Vortex at high speed for about 30 seconds until the pellet is completely resuspended. Incubate with shaking at 30 degrees Celsius for 30 minutes.
Next, add beta me capto ethanol to 0.1 molar incubate with shaking at 30 degrees Celsius for another 30 minutes. After the 30 minute incubation, add two milliliters of boiled and chilled salmon sperm DNA to the cell mixture. Pour the cell mixture into an autoclavable single well plate, and then remove any precipitate that forms because this may interfere with downstream pipetting.
Using the biobot rapid plate aliquot 50 microliters of cell mixture to each well of the four previously prepared. 96 well plates containing plasma DNA do not mix. Incubate the plates at room temperature for 30 minutes without shaking while the cells are incubating.
Prepare 200 milliliters of PEG DMSO lithium acetate solution. Combine 160 milliliters of 50%PEG 33, 50 20 milliliters of DMSO and 20 milliliters of one molar lithium acetate. It is important that this solution is prepared just before use.
Add 125 microliters of PEG lithium acetate DMSO solution to each. Well mix by aspirating and dispensing the cell suspension eight times. Incubate at room temperature for 30 minutes without shaking.
Next, heat shock the cells at 42 degrees Celsius for 15 minutes In a dry incubator, do not stack the plates after the heat shock, spin the plates using centrifuge adapters to accommodate the 96 well plates. After centrifugation, remove the PEG solution by inverting the plates over a waste bucket. Then blot the inverted plates on paper towels.
To remove residual liquid, the cells will remain on the bottom of the wells. Rinse the cells by adding 200 microliters of minimal media to each. Well spin the plates again.
Remove the supernatant by inverting over a waste bucket and blotting on paper towels. Add 200 microliters of minimal media to each well with a rapid plate incubate at 30 degrees Celsius for two days without shaking. After two days, small colonies of cells should begin to form at the bottom of each successfully Transformed.
Well to prepare for the Spatting assay, first dispense 200 microliters of OSE based minimal media into each well of a new flat. Bottom 96 well plate.Next. Mix each well of the transformation plate by aspirating and dispensing the cell suspension eight times with a rapid plate.
Inoculate each well of the raffinose plate with five microliters of cell mixture from the transformation plate, incubate at 30 degrees Celsius for one day. On the following day. Small colonies should be present at the bottom of each.
Well perform the spotting assay in the hood, sterilize a 96 bolt, replicator or frogger by flaming. Mix the colonies with a frogger and then spot colonies on selective media plates. After spotting, allow the plates to dry in the hood for five to 10 minutes.
Incubate the plates at 30 degrees Celsius for Two to three days. Shown Here are representative results of a yeast plasmid over expression screen to identify suppressors and enhancers of TDP 43.Toxicity. TDP 43 is a human protein that has been implicated in the pathogenesis of a myotrophic lateral sclerosis or Lou Gehrig's disease expressing TDP 43 in yeast cells results in aggregation and cytotoxicity.
These plates display colonies with an integrated galactose inducible, T DP 43 plasmid that have also been transformed with plasmids from the flex gene ORF expression library. The colonies here appear red in color because the strain happens to have a mutation that results in accumulation of a red pigment. The plate on the left contains glucose, which represses expression of T DP 43 or the flex gene plasmids.
The plate on the right contains galactose, which induces the expression of TDP 43 and the O Fs in the flex gene plasmids. The green arrowhead indicates a colony transformed with a plasmid that suppresses the toxicity of TDP 43 as indicated by faster growth and more dense colonies. The red arrowhead indicates a colony transformed with a plasmid that enhances the toxicity of TDP 43 as indicated by slower growth and less dense Colonies.
After watching this video, you should have a good understanding of how to transform an avoid library of plasma DNAs de cells.
