This experiment describes experimental methods regarding two-hybrid and other two-component systems. This experiment comes from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Experiments with two-hybrid and other two-component systems
Materials and Instruments
Carriers and Yeasts Move Phase I Identification of Decoy-LexA Fusion Proteins For more product details, please visit Aladdin Scientific website.
Buffers and Solutions SDS Gel Sampling Buffer SDS Polyacrylamide Gel Nucleic Acids and Oligonucleotides Antibodies Culture Media
Specialty Equipment
Materials
Buffers and solutions
Dilute the storage solution to the appropriate concentration.
2XSDS Gel Sampling Buffer
100 mmol/L Tris-Cl (pH 6.8)
200 mmol/L Dithiothreitol
4% SDS (electrophoretic grade)
0.2% Bromophenol Blue
20% Glycerol
SDS Gel Sampling Buffer without dithiothreitol can be stored at room temperature. Add dithiothreitol to 1mol/L storage solution before buffer use.
Gel
SDS Polyacrylamide Gel
To prepare SDS polyacrylamide gels for protein separation, see Appendix 18.
Nucleic Acids and Oligonucleotides
Target DNA encoding the target protein (bait)
Antibodies
Monoclonal antibody against LexA (CLONTECH) or polyclonal antibody against LexA (Invitrogen) or specific antibody against the fusion domain of the target protein (if available).
Medium
Please see Appendix 2 for the composition of the yeast culture medium.
CM Selective Medium
Use Table 18-3 to estimate the amount of medium to be used, and Table 18-4 to prepare the necessary selective medium.
Commercially available yeast nitrogen sources (YNB) without oxalic acid are available with or without amine sulfate. Table 18-4 assumes that YNB contains amine sulfate. If the lighted bottle of Yeast Nitrogen Source indicates that the medium is to be prepared by adding 1.7 g/L, it does not contain amine sulfate and 5 g of amine sulfate per liter of medium should be added.
Yeast Selective X-gal Medium
1. Prepare a basal medium according to Table 18-4 with 900 ml of water. Autoclave the basal medium and cool to 55°C.
2. In a separate bottle, dissolve 7 g of disodium hydrogen phosphate and 3 g of sodium dihydrogen phosphate in 100 ml of distilled water and sterilize.
3. Mix the two autoclaved solutions together and add 0.8 ml of X-gal (in N,N-dimethylformamide) at a concentration of 100 mg/ml and spread the plate.
YPD medium
20 g peptone
10 g yeast extract
20 g glucose
20 g agar (if used in plates)
Add 1L of distilled water and autoclave for 20 min. cool autoclaved medium to 55C before spreading.
Specialized equipment
Dry ice/ethanol bath
See step 13.
Flat-tipped toothpick, sterilized
For sterilization, transfer the standard toothpick to a 250 ml beaker, cover the beaker with aluminum-platinum paper and autoclave under standard dry conditions.
A heating pack or thermal cycler preset to 100°C or a boiling water bath.
Additional reagents
Step 1 of this protocol requires the subcloning reagents listed in Chapter 1, Protocol 17.
Step 2 of this protocol requires the Yeast Transformation Reagent, listed in Spector et al.
Step 17 of this protocol requires the immunoblotting reagents listed in Appendix 8.
Vectors and Yeast
Saccharomyces cerevisiae for vector selection and amplification (see Table 18-6)
Vectors with LexA (see Table 18-1) and activation domain fusion sequences (see Table 18-2), and LacZ reporter plasmids (see Table 18-5). 
Methods
Construction of Decoy-LexA Fusion Proteins
1. Clone the target DNA encoding the decoy protein into the polycomb junction of a LexA fusion vector (e.g., pMW101 or pMW103) to synthesize an in-frame LexA fusion gene. The presence of a translation termination sequence at the carboxyl terminus of the decoy sequence is determined. The resulting plasmid serves as pBait.
2. A series of EGY48 lexAop-LEU2-selected transformed yeasts was established using the following combination of LexA fusion genes and reporter plasmids:
a. pBait + pMW112 (activation assay)
b.pSH17-4+pMW112 (positive control for activation)
c.pRFHMl+PMW112 (negative control for activation)
d.pBait+pJK101 (inhibition/DNA binding assay)
e.pRFHMl+pJK101 (positive control for inhibition)
f.pJK101 alone (negative control for inhibition)
See Tables 18-1 and 18-2 for a description of each plasmid function.
3. Spread each transformation mixture on a suitable selectivity-deficient plate: CM(Glu)-Ura-His (for plasmid combinations a~e) or CM(Glu)-Ura (for plasmid combination f). The plates were incubated at 37°C for 2-3d to select transformed yeast clones containing the plasmid.
If clones do not appear within 3~4d, or if only a very small number of clones (<20) are present, then the yeast should be re-transformed.
4. Prepare master plates of transformants from which specific clones with lacZ and LEU2 reporter activation phenotypes can be analyzed as described in steps 5-9.
Identification of activating and inhibitory activities: analysis of X-gal and Leu2 phenotypes
Steps 5 to 9 were used to test the transcriptional activation of the decoy-LexA fusion proteins and to confirm that the fusions of the decoys do not affect the DNA-binding activity (see Figure 18-9 for a graphical representation of the repression analysis). For each plasmid combination transformed in step 2, several individual clones were selected for analysis. This is important for certain decoy constructs because protein expression levels will vary from clone to clone, just as the apparent ability to activate transcriptional activation of two reporters varies. An alternative to steps 5-8, testing activation by chloroform overlap analysis, is given in the Alternative Scenario at the end of Stage 1.
5. From each transformation of a~f (taken from step 2), select about 8 clones with a sterile flat-tipped toothpick. Touch the clones with a clean toothpick to pick the cells so that they become lcm-long strands in small compartments on a fresh CM(Glu)-Ura-His or CM(Glu)-Ura plate. Usually up to 60-80 lines can be formed on a single plate. Incubate the plates at 30°C overnight.
6. The next day, redraw lines from the two mother plates onto each of the following plates:
Transformations a~f: streak onto CM(Glu,X-gal)-Ura and CM(Gal,X-gal)-Ura
Transformations a~c: line to CM(Glu)-Ura-His-Leu and CM(Gal)-Ura-His-Leu 
7. Incubate plates at 30°C for 4d.
8. analyze for inhibition and activation;
a. For inhibitory activity, observe the X-gal phenotype at approximately 12 to 24 h after streak inoculation.
b. For activation, observe X-gal phenotype between 18 h and 72 h of lineage inoculation.
c. For activation, observe the Leu2 phenotype between 48 and 96 hours. The expected results for a well-performing bait are given in Table 18-7 and are summarized below.
(1) Preferably between 2 and 24 h after inoculation with CM(Gal,X-gal)-Ural, it should be possible to see that the d+e transformation is lighter in color than f. The d+e transformation should be observed from 48 h to 96 h.
At 48 h of inoculation into CM(Glu,X-gal)-Ura, the b transformation should be bright blue, c should be white, and a should be white or a very light blue color.
(2) At 48 h after inoculation, on CM(Glu)-Ura-His-Leu or CM(Gal)-Ura-His-Leu, the b transformation should grow as well as on the CM(Glu)-Ura-His mother plate, while a and c should not grow.
(3) Ideally, the a-transformation should not show significant growth for 96 h after inoculation. 
9. Based on the results of the inhibition and activation analyses, select the appropriate candidate clones. 
Detection of decoy protein expression
10. On the master plate, mark the clones to be analyzed for protein expression. Use clones that have been shown to be suitable for expression of the decoy as basidiomycete culture for library transformation (in Phase II).
11. For each new bait construct, analyze at least two primary transformants. Also include two transformants that serve as positive controls for protein expression (e.g., pRFHMI).
The clones should be grown in CM(Glu)-Ura His liquid medium. a. Use a sterile toothpick to pick clones from the CM(Glu)-Ura-His master plate. b. Use a sterile toothpick to pick clones from the CM(Glu)-Ura-His master plate. If gloves are worn, the toothpick can be dropped into the culture tube and left there without fear of contamination.
b. Incubate overnight at 30°C on a tumbler or other shaking apparatus.
c. In the morning, dilute the saturated culture solution into a new culture tube containing 3-5 ml of CM(Glu)-Ura-His to give an initial density OD600 of about 0.15. Incubate at 30°C for 4-6 h until the optical density is approximately tripled ( OD600 of about 0.45 to 0.7).
12. Transfer 1.5 ml of culture medium to a microcentrifuge tube and centrifuge the cells at maximum speed for 3~5 min. the volume of visible precipitate should be 2~5 ul. carefully decant or aspirate the supernatant.
Some, though not all, LexA fusion proteins exhibit a dramatic increase in detectable protein levels with the initiation of growth stationary phase. Thus, it is not necessary to saturate the culture with the expectation of increasing the yield of analyzed proteins.
If more than one round of analysis is anticipated, it may be advantageous to freeze duplicate samples at this stage.
13. Add 50ul of 2XSDS Gel Spiking Buffer to the centrifuge tube and shake the tube rapidly to suspend the precipitate. Immediately place the centrifuge tube on dry ice or in a dry ice/ethanol bath.
The sample may be used immediately for analysis or frozen at -70°C so that it remains stable for at least 4 to 6 months.
14. Transfer the sample directly from dry ice or -70°C to 100°C and boil for 5 min.
PCR set to 100°C is the most convenient, but of course a water bath or heat can also be used.
15. Cool the sample on ice and centrifuge at maximum speed for 5-30s to precipitate large cellular debris. Add 20-50ul of sample to each lane of the SDS polyacrylamide gel.
16. Electrophoresis and analyze the products to determine if the decoy proteins of the expected size are expressed at reasonable levels.
17. To prevent possible problems, analyze yeast lysates containing LexA-fused decoys by immunoblotting (see note on this step).
The immunoblot can be performed as described in Appendix 8. LexA fusions can be detected with an antibody against the fusion domain or, if this is not available, an anti-LexA antibody can be substituted.
An important step in identifying a decoy protein is to directly analyze whether the decoy is detectable and whether the decoy is the correct size. In most cases, both of these are correct. However, some proteins (especially with fusion domains at ~60~80kDa or larger) are either synthesized at very low levels or are proteolytically sheared after translation. Both of these results can lead to problems in library screening. Proteins that are expressed at low levels and have no apparent activity in transcriptional activation analyses can be upregulated to high levels under leucine selection and then suddenly show a high background of transcriptional activation. In the case of proteins that are sheared due to the occurrence of proteolytic hydrolysis, screening is still possible for LexA fused only to the amino terminus of a larger bait. 
Phase II Screening for an Interactor
Material
Buffers and solutions
Dilute the storage solution to the appropriate concentration
Dimethyl sulfoxide (DMSO)
Ethanol
Optional, see step 9.
Sterile glycerol solution for freezing transformants
65% sterile glycerol
0. 1mol/LMgS04
25 mmol/L Tris-Cl (pH 8.0)
TE (pH 7.5) (sterile)
TE(pH7.5) with 0.1 mol/L lithium acetate
TE(pH7.5), containing 40% PEG4000 and 0.1mol/L lithium acetate (sterile)
Nucleic acids and oligonucleotides
Vector DNA
Sheared salmon sperm DNA is typically used as a carrier. High quality DNA is very important. The use of low quality products may reduce the frequency of transformation by 1 to 2 orders of magnitude. For a simple method of producing high quality salmon sperm DNA, see the method of Schiestl and Gietz (1998) and Scheme 10 in Chapter 6, or choose one of the commercial products sold by many companies.
Interaction screening libraries
Medium
CM Selection Medium
Use Table 18-3 to estimate the amount of medium required and prepare the required selection medium according to Table 18-8.
Amino acid-free yeast (YNB) is available with or without ammonium sulfate. Ammonium sulfate is assumed to be present in YNB in Table 18-8. If the Yeast Nitrogen Source bottle states that the medium is to be prepared with 1.7 g/L Yeast Nitrogen Source, then it does not contain ammonium sulfate and should be prepared with 5 g of ammonium sulfate per liter of medium.
Yeast Selection X-gal Medium
1. Prepare a basal medium according to Table 18-8 in 900 ml of water, autoclave, and then cool to 55°C.
2. In a separate bottle, dissolve 7 g of disodium hydrogen phosphate and 3 g of sodium dihydrogen phosphate in 100 ml of distilled water and autoclave.
3. Mix the two autoclaved solutions together, add 0.8 ml of X-gal (grated in N,N-dimethylformamide) at a concentration of 100 mg/ml, and spread the plates.
Centrifuge and rotor
Sorvall GSA rotor or equivalent
Sorall RT6000 centrifuge and H1000B rotor or equivalent
Specialized equipment
Culture poles (24 cmX24 cm) for selective media (see Table 18-3)
These plates are very expensive, but can be reused many times (see step 9).
Falcon tubes (50 ml, sterile)
Glass beads (0.45 mm diameter, sterile; Sigma)
Optional, see step 9.
Heating block, preset to 42°C
Microtitration plate (96 wells)
See step 21.
Multichannel pipettes or inoculation multitubes/frogs (e.g. Dankar Scientific)
Optional, see step 21.
Frogs for multicolony transfer can be purchased or easily manufactured; it is important that all spokes of the frog have a flat surface and that the ends of the spokes are level. Frothers can be sterilized by autoclaving or ethanol cauterization.
Carriers and Bacterial or Yeast Strains
Candidate strains of Saccharomyces cerevisiae carrying vectors expressing bait proteins and the lexAop/lacZ reporter (from Phase -). 
Methods
Transformation libraries
1. A yeast colony expressing the bait protein and the lexAop-lacZ reporter in the best condition in the original control assay in phase I was selected and inoculated in 20 ml CM(Glu)-Ura-His liquid medium and incubated overnight at 30°C with shaking.
IMPORTANT: The bait protein and lexAop-lacZ reporter plasmid should be transformed into yeast within 7~10d prior to retransformation with the library. It is important to maintain sterile conditions at all times throughout the experiment.
2. Dilute 20 ml of the overnight culture in 300 ml of CM(Glu)-Ura-His liquid medium so that the OD600 of the diluted culture is about 0.10~0.15. Shake the culture at 30°C on a rotary shaker until the culture proliferates 1~5-fold and the OD600 reaches 0.50.
3. Transfer the culture to a sterile 250 ml centrifuge bottle and centrifuge at 1000~1500 g (2500~3000r/min Sorvall GSA rotor) at room temperature for 5 min. Remove the supernatant, add 30 ml of sterile water, gently tap the bottle on the bench to re-suspend the precipitate, and transfer the mixture to a 50 ml sterile Falcon tube. Transfer the mixture to a sterile 50 ml Falcon tube.
4. 1000~1500 g (using Sorvall GSA rotor 2500~3000r/min) centrifuge the yeast cells for 5 min. pour off the water and re-suspend the yeast cells in 1.5 ml of TE (pH 7.5) containing 0.1mol/L lithium acetate.
5. Add 1ug of library DNA and 50ug of freshly denatured vector DNA to each of 30 sterile 1.5 ml microcentrifuge tubes. immediately add 50ul of yeast suspension (from step 4) to each microcentrifuge tube.
-For a successful transformation assay, approximately 105 transformants should be produced per microgram of library DNA. The use of small volumes of DNA (preferably less than 10ul per tube) usually improves transformation efficiency.
Multiple small volumes of yeast transformed in parallel are good for minimizing the probability of contamination, and this method often results in significantly higher transformation efficiency compared to using larger volumes in fewer tubes. Do not add too much transformed library DNA to each suspension of susceptible yeast cells, otherwise each susceptible cell may take up more than one library plasmid, complicating later analysis.
6. Add 300ul of sterile TE (PH7.5) containing 40% PEG4000 and 0.1 ml/L lithium acetate to each tube of cell suspension, gently turn the tubes several times to make the mixture (do not oscillate), and incubate at 30°C for 30~60 min.
7. Add 40ul of DMSO to each tube, turn to mix the suspension, and heat at 42°C for 10min on a heating block.
8. Plate the transformation mixture as follows:
28 of the tubes are for transformant production only
a Add the mixture from each tube to a 24 cmX24 cm CM(Glu)-Ura-His-Trp Selection Plate.
b. Spread the cells well and incubate the plates at 30°C until colonies appear.
Each 24 cmX24 cm plate requires 250-300 ml of medium and should be dried at room temperature for 1-2d after pouring before use. To minimize the chance of contamination, the surface of the plate should be baked over a fire after pouring. Some researchers have adopted a different treatment method: open the plate, turn the lid over and place it in a standard tissue culture ultra-clean bench and expose it to UV light for about 10 min.
The remaining 2 tubes are used to evaluate transformation efficiency
a. From each tube, dispense 350ul of the mixture onto a 24 cmX24 cm CM(Glu)-Ura-His-Trp wrench.
b. Spread the cells and incubate the plate at 30°C until colonies appear.
c. Aspirate the remaining 40ul of mixture from each tube and make a series of 1:10 dilutions (at least 3) with sterile TE (pH 7.5) or water.
d. Aspirate 100ul of each dilution onto a 100 mmCM(Glu)-Ura-His-Trp plate and incubate the plate at 30°C until colonies appear.
Analyzing the dilution gives a rough estimate of the number of transformants obtained. The predicted increase in the number of transformant colonies on the 100 mm indicator wrenches was occasionally significantly different from that on the 24 cmX24 cm plates, especially between batches of wrenches. A successful transformation experiment should yield 20,000-40,000 colonies per large plate.
Harvesting and enrichment of primary transformants
Steps 9~14 provide a frozen stock representing the full set of primary transformants that can be used for later screening. In this experiment, homogenous cell suspensions are prepared from the primary transformants ( ~106 cells), each of which can be used for plate screening in selection medium. This technique represents a more conventional method of transferring yeast (e.g., replicate plate culture), which transfers such a large number of cells that cell growth that somehow results in a spurious background can be observed on the selection medium. If visible mold spots or other contaminations are observed on the plate, then the contaminants and their surrounding parts are carefully cut away with a sterile blade before harvesting the library transformants.
In step 9, the first method (the motif method) operates quickly and allows for induction of the library and screening on the selection medium to be performed on the same day; it also shortens the time the plate is open, thus avoiding contamination originating from airborne molds and bacteria. Approximately one-third of the yeast cell suspension will remain on the plate; however, the amount used normally does not exceed 2% of the collected suspension, so it is important to ensure that yeast colonies elute from the plate with approximately equal probability. The second method in Step 9 (the scraping method) saves reagents and can be easily used on plates that have been cut free of mold and contaminants.
9. Harvest the library using one of the following methods.
1) Harvesting the library by the shaking method
a. Pour 10 ml of sterile water and about 30 sterile glass beads onto 5 separate 24 cmX24 cm plates containing transformants.
b. Stack the 5 plates on top of each other, hold the plate firmly and shake the plate vigorously until the colonies are resuspended (1-2 min).
c. Pipette 5 ml of yeast suspension from each plate with a sterile pipette (tilt the plate) and concentrate the cell suspension in a 50 ml sterile conical tube.
d. Continue with the next 5 plates and repeat steps a to c. Harvest yeast cells from 30 consecutive plates, resulting in a total volume of 150 ml of yeast suspension, which is stored in 3 50 ml tubes.
The same glass beads can be transferred to new plates or new glass beads can be used. More than 5 plates can be eluted at the same time, as long as they can be held in the hand and shaken.
Plates can be reused many times. After harvesting the yeast cells, discard the remaining agar, rinse the plate, wipe with ethanol, expose to UV light for 10 min, and store.
2) Harvesting libraries by scraping
a. Wearing gloves, place 30 24 cmX24 cm plates containing transformants at 4°C to allow the agar to harden (usually 2 h or even overnight).
b. Soak a microscope slide in ethanol and then cauterize it to sterilize it. Use this slide to gently scrape yeast cells from the transformed plate into a 50 ml conical tube. Continuously re-cauterize the slide or replace it with a new one (every 5-10 plates).
Cells from all 30 plates are usually pooled in one or two 50 ml conical tubes.
Plates can be reused many times. After harvesting the yeast cells, discard the remaining agar, rinse the plate, wipe with ethanol, expose to UV light for 10 min, and store.
10. If desired, add sterile TE (pH 7.5) or sterile water to 40-45 ml to each conical tube containing yeast cells, and suspend the cells by shaking or turning the tube.
11. Centrifuge the tubes in a benchtop centrifuge at 1000~1500 g (using Sorvall H1000B rotor at 2200~2700r/min) for 5 min at room temperature and discard the supernatant.
12. Repeat steps 10 and 11.
After the second round of washing, the total volume of cell sediment derived from 1. 5X106 transformants should be 25 ml.
13. Resuspend the tightly packed cell sediment in double the volume of sterile glycerol solution, combine the contents of the different tubes and mix thoroughly.
14. Transfer 1 ml of the cell mixture to a series of small sterile centrifuge tubes and freeze at -70°C (cells remain stable for at least 1 year).
If plate culture is performed directly on selective medium (5 h to complete), leave one portion unfrozen and perform the following steps. Assuming 100% viability of the unfrozen culture, - in general, yeast frozen for less than a year will be expected to have a viability greater than 90%. In case of unforeseen circumstances (e.g., no colonies are obtained, especially when freezing cells in plate culture on selective medium), measure the viability of the frozen cells by making a series of limited dilutions on CM(GlU)-Trp-His-Ura.
Screening for interacting proteins
Each copy of the transformed library was spread on leucine-deficient selection medium to test whether it could promote LEU2 transcription. Not all cells containing interacting proteins grew at 100% efficiency on leucine-deficient selective medium plates (Estojaketal.1995). To increase the probability of detecting the interaction, each clone obtained in the transformation library should appear as 3 to 10 viable yeast cells on the selective culture plate. For example, if 5x105 clones are initially obtained, 1.5x106 to 5x106 cells should be spread on 2 to 5 plates. Although this may result in too much isolation of the same cDNA, it helped us to ensure that all primary transformants were presented with at least 1 cell on the selective media plates. In fact, repeated isolation of an identical cDNA in a relatively small number of positive clones can be seen as a marker of a specific protein interaction.
15. Thaw one copy of the transformant library yeast cells (from step 14), and induce transcription of the GAL1 promoter in the library by incubating the yeast cells with CM(Gal-Raff)-Ura-His-Trp medium at a dilution of 1:10 and shaking at 30°C for 4 hours.
16. 106 cells were cultured on the appropriate number of 100 mm CM(Gal-Raff)-Ura-His-Trp-Leu dropout plates (or 50ul OD 600 for 1.0 culture).
Important: 105 cells per plate is the highest plate density that is usually effective. Higher plate densities (e.g. 3x106) may result in yeast cell cohabitation, leading to high background growth.
17. Incubate plates at 30°C for 5d.
Depending on the individual bait proteins used, good candidates for positive interactors usually grow clones within 5d, with most clones appearing within 4d.
18. Observe the growth of the plates and mark clones as they appear.
A good strategy (especially if a large number of clones are growing) is to observe the plate every day, and it is not necessary to pick clones immediately. Rather, the first day's clones that are visible to the naked eye should be marked on the plate with a lab marker with a specific color dot (e.g., red on day 3). Each day the newly grown clones are marked with a different color pen (blue on day 4, etc.).
19. On the fifth day, form a master plate grouped by the different clones that appear each day (CM[Glu]-Ura-His-Trp). If many distinct positive clones appear, it will be necessary to generate a number of individual plates according to the clones appearing on the second, third, and fourth days, respectively. To make a negative control for later steps, pick 3 to 5 clones at random from the plate on which yeast cell viability was assayed (see instructions for step 14) and incubate them in parallel lines on a test master plate. If multiple tubes/frogs are used in later steps of the experiment, be sure to line the appropriate grid to ensure that the colonies correspond to the spokes on the frog (see Figure 18-10).
Incubate plates at 20.30°C until spots/colonies form. 
Initial Determination of Positive Interactions: β-Galactosidase Activity and Leucine Requirement Assays Steps 21 and 22 detect galactose-induced transcriptional activation of the lexAop-LEU2 and lexAop-lacZ reporter genes. The simultaneous expression of both reporter even genes under conditions of galactose-specific induction suggests that this transcriptional phenotype occurs because of the expression of the library-encoded proteins and not because of a mutation in the yeast host bacterium. Master plates containing glucose and leucine can be used to provide test clones.
21. determination of transcriptional activation.
This experiment can be performed using either a toothpick restriping culture or by utilizing a multibranch/frogator, which is useful when analyzing a large number of positive clones (see Figure 18-10 for details on frogators).
1) Determination by direct line culture
a. Use a flat-tipped toothpick to duplicate the same grid on each of the 4 plates below as on the main plate. Use the same toothpick to line culture the monoclonal clones on each of the four plates, trying to make as thick a yeast line as possible on the X-gal containing plate and a thin yeast line on the leucine deficient plate.
CM(Glu/X-gal)-Ura-His-Trp 1 plate
CM(Glu/Raff/X-gal)-Ura-His-Trp 1 plate
CM(Glu)-Ura-His-Trp-Leu 1 plate
CM(Glu/Raff)-Ura-His-Trp-Leu 1 plate
b. Plates were incubated at 30°C for 3~4d.
X-gal plates yielded results within 48 h, with the strongest interactions growing spots even at 18 h. Different growth conditions on leucine plates between 48 and 72 h were often evident.
2) Measurement with multiple tubes/frogs
a. Add 25-30ul of sterile water to 48 wells of a 96-well microtitre plate.
b. Use a frottage device to transfer the plaque from the pre-drawn grid on the main plate to the wells of the microhalo titration plate at the same time. Gently shake the titration plate.
c. Use the frottage again to transfer the yeast from the microtitre plate to the plate below. This method transfers approximately the same number of cells to the same plate.
CM(Glu)-Ura-His-Trp 2 plates
CM(Gal)-Ura-His-Trp 1 plate
CM(Glu)-Ura-His-Trp-Leu 1 plate
CM(Gal/Raff)-Ura-His-Trp-Leu 1 plate
d. Pl