Feb 10, 2026
semi-quantitivate Yeast Two-Hybrid (sqY2H)
- Florent Laval1
- 1Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA
Protocol Citation: Florent Laval 2026. semi-quantitivate Yeast Two-Hybrid (sqY2H). protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6eknzgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: December 15, 2025
Last Modified: February 10, 2026
Protocol Integer ID: 235072
Keywords: missense variants on protein, protein interaction, yeast, protein, missense variant
Funders Acknowledgements:
National Human Genome Research Institute (NHGRI)
Grant ID: UM1HG011989
Abstract
Assessment of the impact of missense variants on protein-protein interactions
Troubleshooting
Cloning into yeast two-hybrid vectors
LR reaction
- Put all plasticware (PCR plates, reservoir, tubes) On ice before starting.
- All reagents should be thawed On ice , thoroughly vortexed and briefly spun to remove any liquid from the cap.
- Assemble the following master mix:
| Reagent | Stock concentration | Final concentration | Volume (uL)/reaction | Volume (uL)/100 reactions | |
| TE buffer, pH 8.0 | / | / | 1 | 100 | |
| Gateway™ LP Clonase™ II Enzyme mix | 10x | 1x | 1 | 100 | |
| pDEST-DUAL | 100 ng/uL | 20 ng/uL | 1 | 100 |
LR master mix.
- Vortex the master and transfer into a pre-chilled reservoir.
- Aliquot 3 µL of the master mix into a new PCR plate using a automatic multichannel pipette.
- Using a multichannel pipette, add 2 µL of the purified entry products to the plates preloaded with the LR master mix.
- Seal the plates and spin 00:05:00 at 1000 rpm .
- Incubate the plates at 25 °C Overnight . After incubation, BP reaction plates can be stored at -20 °C .
Transformation into DH5a cells
- Put all plasticware (PCR plates, reservoir, tubes) On ice before starting.
- Thaw E. coli DH5a cells On ice .
- Gently mix tube by inverting (do not vortex!) to homogenize the suspension.
- Transfer cells to a pre-chilled reservoir and aliquot 10 µL of cells into each well of a pre-chilled empty PCR plate using an automatic multichannel pipette.
- Add 3 µL of BP product into the cell suspension using a multichannel pipette.
- Mix the cells by swirling the pipette tips in the wells after dispensing.
- Incubate On ice for 00:30:00 .
- Using a 96well heating block, pre-heated at42 °C , heat-shock the cells for 00:00:45 .
- Immediately return the cells On ice for 00:02:00 .
- Carefully add 90 µL of complete SOC media to each well using an automatic multichannel pipette.
- Seal the plate with an airpore tape and incubate at 37 °C for 01:00:00 .
- Fill a costar plate with 120 µL of LB media containing 100 µg/mL ampicillin.
- Transfer 10 µL of the recovered cells into the costar plates and seal with airpore tape.
- Incubate at 37 °C Overnight .
Plating and spreading of bacterial culture
Liquid bacterial cultures are spread onto solid selective media to isolate single colonies.
This was conducted on 15 cm diameter petri dishes with a Tecan Evo robot, using a custom program.
Importantly, glycerol stocks of the liquid cultures are generated, in two copies, by combining 40 µL of 40 Mass Percent sterile glycerol and 40 µL of overnight culture. These glycerol stocks are sealed with aluminum foil and stored at -80 °C .
Colony picking and isolation
- Fill costar plates with 150 µL LB media supplemented with 100 µg/mL ampicillin.
- One colony is picked and isolated by hand with sterile wooden toothpicks from each spot on solid media and transferred into the costar plates prefilled with LB+ampicillin.
- Seal plates with airpore tape and incubate at 37 °C Overnight .
- The next day, make glycerol stocks, in three copies, by combining 40 µL of 40 Mass Percent sterile glycerol and 40 µL of overnight culture.
- Seal plates with aluminum foil and store at -80 °C .
DNA purification
- Fill deep well plates with 1.2 mL LB media supplemented with 100 µg/mL ampicillin.
- Inoculate 10 µL glycerol stocks.
- Seal plates with airpore tape and incubate at 37 °C Overnight .
- The next day, spin for 00:15:00 at 2000 rpm , or until cells form a pellet.
- Discard supernatant.
- DNA is extracted using a QIAGEN BioRobot 8000.
Plasmid pool sequencing
To confirm the successful transfer of the desired DNA insert from the entry vector into the destination vector, all purified DNA samples are sequenced. A pooled sequencing strategy is employed on the Illumina platform, where each pool contains one clone from different genes to ensure reliable identification.
Preparation of DNA pools
- Measure the concentration of each 96-well (containing DNA pool).
*For this step, a Qubit dsDNA BR Assay Kit (catalog: Q32851) suffices.
- For each well, take an aliquot and dilute with PCR grade water in a new 96-well plate (label “sample plate”) to the required concentration of 15 ng/µL
Nextera Oligo Setup
Pre-mix i5 and i7 oligos for a concentration of 10 micromolar (µM)
Illumina Tagment MasterMix prep
| Reagent | 1X (single reaction) | 100X (one full plate) | |
| Tagment DNA (TD) buffer | 2µL | 200µL | |
| PCR grade water | 6.5µL | 650µL | |
| Tagment DNA Enzyme (TDE1; catalog: 20034197) * | 0.25µL | 25µL |
*Keep @ -20 °C , or on ice when in use!
- Use a multichannel pipette to add 8.75 µL MasterMix to each well of a 96-well plate (label “reaction plate”)
- Use a multichannel pipette to add 1.25 µL DNA (15 ng/µL ; from the sample plate) to each corresponding well of the reaction plate
- Gently pipette to mix
- Cover the reaction plate with a BioRad PCR plate seal
- Incubate the reaction plate for 00:10:00 @ 55 °C in a thermal cycler
- After, immediately place the reaction plate on ice
- Proceed immediately with the PCR
PCR amplification of tagmented DNA
Using a new 96-well plate (labeled amplification), add the following:
| A | B | |
| Reagent | 1X (per well) | |
| 2X Phusion MasterMix (catalog: M0530S) | 10µL | |
| i5 & i7 oligo (10uM; see above/attached sheet) | 2µL | |
| Tagmented DNA | 8µL |
- Using a multichannel pipette, pipette gently to mix
- Cover with a BioRad plate seal and placed in a thermal cycler
| Cycle(s) | Time | Temp | |
| 1 | 3min | 72°C | |
| 10 | 10sec | 98°C | |
| 30sec | 56°C | ||
| 3min | 72°C |
Thermal cycler Program
- Check 10 random wells confirming that the PCR worked by running 2 µL of each on a 1% EX e-gel along with a 1kb ladder.
- See a smear from ~100bp to ~5kb and each lane should have roughly equal amounts of product.
Pool amplified DNA
Using a multichannel, pool all wells (10 µL from each well)
Purifying for sequencing
- Vortex to mix the pooled, amplified DNA
- Load all wells of a single 1% EX e-gel from ThermoScientific (one plate per gel; catalog: A42100)
- Run using 50V until the 100bp band (1kb+ DNA ladder, catalog: N0469S) reaches the bottom of the gel.
- Cut the smear from 500bp-800bp
- Purified using the Qiagen gel purification kit (catalog: 28604)
- Elute the library in 15 µL
- Measure concentration using Qubit dsDNA HS Assay Kit (catalog: Q33230)
Yeast transformation
Haploid S. cerevisiae strains Y8800 (MATa) and Y8930 (MATα) are transformed with pDest-AD-prey vectors (carrying the TRP1 gene) and pDest-DB-bait vectors (carrying the LEU2 gene), respectively.
Yeast cells are transformed following high-efficiency lithium acetate –
polyethylene glycol protocol – salmon sperm carrier DNA (Yu et al., Science, 2008), that
was modified and adapted to a 96-well format for enhanced throughput. For each
transformation reaction,100 µL of exponential phase yeast cell culture (OD at 600
nm ~0.6-1.0) and 100 ng of high-purity plasmid are used. Yeast cells are plated
onto appropriate solid media (Yu et al., Science, 2008), depending on the selective marker carried by the
plasmid, to select for transformants. Transformants were picked after 120:00:00 of
incubation at 30 °C .
sqY2H
Experimental rearraying of the strains
- Inoculate the yeast strains into 200 µL appropriate selective media in round bottom plates and incubate at 30 °C for 48:00:00
- After this incubation period, the yeast strains are re-arrayed with a Tecan Genesis robot, using a customized protocol
- Cell are incubated at 30 °C for 72:00:00 .
- After incubation, glycerol stock are made in two copies, by combining 40 µL of 40 Mass Percent sterile glycerol and 40 µL of culture.
- The rest of the culture is used in the mating step
Mating
- Dispense 200 µL YPD into round bottom plates
- Combine equal volumes of the corresponding rearrayed DB and AD cultures (5 µL each) into these prefilled plates
- Seal the plate with an airpore tape and incubate at 30 °C for 24:00:00 .
Diploid selection
- Dispense 200 µL SC-LW into round bottom plates
- Transfer 10 µL mated culture
- Seal the plate with an airpore tape and incubate at 30 °C for 48:00:00
Interaction selection
- 5ul of diploid selection cultures are spotted in quadruplicates onto the following selective media:
1. SC-LW: selection for diploid cells. This controls for the presence of diploid cells harboring both AD and DB plasmids.
2. SC-LWH + 1 millimolar (mM) or 10 millimolar (mM) or 25 millimolar (mM) 3-Amino-1,2,4-triazole (3-AT)
- Let the plates dry at Room temperature for 01:00:00
- Move the plates to 30 °C and incubate for 120:00:00 .
- Take pictures at day 3, day 4 and day 5.
Scoring
- Pictures are scored with the CellProfiler software, using a customized script
Sequencing confirmation using SWIM-Seq
Lysates
In addition to being spotted on solid selective media, cultures of diploid yeast cells are processed as previously described (Dreze et al., 2010) to generate cell lysates
Duplex PCR
PCR is conducted on each lysate sample to amplify the DB-X and AD-Y fusion constructs.
Barcoded primers are used for AD and DB primers, to assign well identity to the amplicons
- Thaw the primers and the diluted miniprep DNA plates at room temperature, vortex, and spin
quickly to remove liquid from the lid.
- In a tube On ice , assemble a master mix as described in substep 13.1.
- Aliquot 29 µL of the master mix with an automatic multichannel pipette into the PCR plates.
- Using a 12 channel pipette, distribute 1 µL of the lysates to the PCR plates preloaded with master mix.
- Seal plates, vortex and spin 00:05:00 at 1000 rpm .
- PCR is conducted for 35 cycles, using the following cycling conditions:
| A | B | |
| 98 °C | 5 min | |
| 98 °C | 30 sec | |
| 57 °C | 30 sec | |
| 72 °C | 5 min | |
| 72 °C | 10 min | |
| 10 °C | Hold |
- After PCR, spin plates quickly to remove droplets from the lid.
- The first row of each PCR plate is run on a 1% agarose gel to check for successful DNA amplification
- 96well plates are pooled into a single well
- Pooled samples are purified using a QIAquick 96 PCR Purification kit from Qiagen
- During sequencing library preparation, Illumina adapter sequences, i7 and i5, were incorporated as SWIM plate indices. The library was then sequenced using an Illumina platform (MiSeq or NextSeq 500).
PCR master mix
| Reagent | Stock concentration | Final concentration | Volume (uL)/reaction | Volume (uL)/100 reactions | |
| Dream Taq HS Mix | 2x | 1x | 15 | 1500 | |
| Primer AD | 10 uM | 0.2 uM | 0.6 | 60 | |
| Primer DB | 10 uM | 0.2 uM | 0.6 | 60 | |
| Primer term | 10 uM | 0.2 uM | 0.6 | 60 | |
| Water | / | / | 12.2 | 1220 |
PCR master mix
Protocol references