This MY two H system improves on the conventional yeast two hybrid protocol to markedly reduce the number of false positives. First, prepare a small scale transformation of the bait plasmid into yeast. After validating the bait construct using a competitive inhibitor screen the cDNA library on minus four plates.
Proceed to verify positive clones with XGL and reation assays. Then identify the potential interacting proteins by DNA sequencing and bioinformatics analysis. Ultimately, this modified yeast two hybrid system significantly increases efficacy and success of the screen process.
The conventional yeast two hybrid system is notorious for generating force positives. This message show the modified screen procedure that is significantly improved in favor of two point tails. So let's get started.
Inoculate 50 milliliters of YPD with a five milliliter overnight culture of MAV 2 0 3. Grow two to four hours at 30 degrees Celsius. Then harvest the yeast by centrifugation after one wash resuspend pellets in two milliliters of solution one and incubate at room temperature for 10 minutes.
Prepare the DNA mix for transformation. Add 100 microliters of yeast cells and mix well. Then add 700 microliters of solution two and mix well.
First incubate at 30 degrees Celsius for 30 minutes, and then heat shock. The cells now harvest cells by centrifugation and resuspend the pellet in 200 microliters autoclave distilled water spread serial dilutions of the suspension onto SD LU plates incubate at 30 degrees Celsius for two to three days. This step confirms that the bait plasmid activates transcription and that this self activation can be neutralized by inhibitors like three at a competitive inhibitor of the hiss three gene product.
First, transform the bait plasmid into MAV 2 0 3 yeast. Spread the reaction on SD LU plates and incubate at 30 degrees Celsius. In order to screen colonies for three at induced self activation, use autoclave toothpicks to patch colonies from each transformation onto SD LU hiss plates containing increasing concentrations of three at incubate plates at 30 degrees Celsius.
Eliminate bait strains that grow on plates containing 100 millimolar three at as not suitable for use in the two hybrid screen for re. For remaining baits, select the lowest three at concentration that inhibits cell growth to use in CD NA library screening, suspend several isolated colonies of yeast containing the bait plasmid in 100 microliters of autoclave distilled water. Spread them onto two 10 centimeter SD LU plates.
Incubate both plates at 30 degrees Celsius. Scrape and completely suspend the cells in 10 milliliters. Autoclave distilled water and inoculate 500 milliliters of liquid YPD.Medium.
Verify cell density by spectra, photometry, and culture at 30 degrees Celsius. Proceed to harvest cells by centrifugation. Wash once in 100 milliliters, autoclave distilled water, and once in 50 milliliters of solution one.
Then resuspend the pellet in 2.5 milliliters solution one. Now add denatured sheared salmon sperm DNA, and also the CD NA library mixed gently by pipetting. Then add 15 milliliters of solution two containing 40%polyethylene glycol.
Mix gently and distribute 700 microliter aliquots into 25 autoclave. 1.5 milliliter micro centrifuge tubes incubate for 30 minutes in a 30 degree Celsius water bath, followed by heat shock of 15 minutes. In a 42 degree Celsius water bath.
Harvest the cells by centrifugation gently resuspend each pellet in 400 microliters autoclave distilled water. Spread each transformation mixture onto two selection plates and incubate for five to 10 days at 30 degrees Celsius as a control estimate of the transformation efficiency of the reaction plate. Serial dilutions of one reaction.
After incubating for three days at 30 degrees Celsius, determine the number of total colonies. Transfer the colonies onto a YPD plate and incubate at 30 degrees Celsius overnight. Then place a rounded nitrocellulose membrane directly on the YPD plate, ensuring no air bubbles.
After a minute, check that all colonies are transferred to membranes. Position the membrane colony side up in an aluminum foil boat and place it on the surface of liquid nitrogen for 20 seconds. Then immerse in the liquid nitrogen for a couple of minutes.
Now remove the membrane to thaw. In the meantime, prepare a Petri dish with fresh XCA solution layer Aman paper. Then carefully immerse the thawed membrane and incubate at 37 degrees Celsius.
Until blue colonies appear. The reation of prey, clones, and bait construct into yeast can further eliminate false positives. First, isolate plasmid DNA from the yeast strains potentially containing interacting proteins.
Transform DNA into e coli DH five alpha and select by growth on LB ampicillin plates. Screen transformants by restriction enzyme analysis. Now co transform the prey and bait plasmids into yeast MAV 2 0 3 plate on the SC LU trip plates and incubate for two to three days at 30 degrees Celsius.
Pick three different colonies to perform the xal assay sequence, the plasmid DNA from the positive clones and identify genes using bioinformatics. In this experiment. The Rine growth factor pro Granulin was used as bait to screen a brain CD NA library 54 positive clone.
Candidates were isolated from among 2.5 million. Transformants screened with the pro granulin bait. Further analyses by the ex skull assay indicated 23 positive clone candidates for the pro granulin bait.
Interestingly, bioinformatic analyses of sequencing data identified TNF R two.Indeed. Further functional experiments confirmed progranulin as a novel ligand of TT NF receptors. After watching this video, you should have a good understanding of how to perform this modified used hybrid screen with your base of interest.
Don't forget to work with gloves as some regions used are hazards. Good luck with your experiments.
