Feb 11, 2026
Validation in mammalian NanoLuc Two-Hybrid
- 1Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA
Protocol Citation: Florent Laval 2026. Validation in mammalian NanoLuc Two-Hybrid . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzem8rvx1/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 11, 2026
Protocol Integer ID: 235074
Keywords: missense variants on protein, validation in mammalian nanoluc two, mammalian nanoluc two, protein interaction, missense variant, protein
Funders Acknowledgements:
National Human Genome Research Institute (NHGRI)
Grant ID: UM1HG011989
Abstract
Assessment of the impact of missense variants on protein-protein interactions
Cloning into N2H 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 | |
| Destination vector | 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) (see attached sheets)
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)
mN2H
2d 0h 30m 2s
Cell plating
- Seed HEK293T cells at 54,000 cells in 100 µL in a 96-well white Greiner plate
- Incubate at 37 °C 24:00:00
1d
Transfection
- Prepare a DNA mix of 100 ng of each partner and up to 12 µL of DMEM per well
- Prepare a PEI mix with 0.6 µL of PEI per well and 7.4 µL of DMEM per well
- Mix PEI and DNA mixes and incubate at Room temperature for 00:30:00
- Add the 20 µL of DNA/PEI mix to each well
- Incubate at 37 °C for 24:00:00
1d 0h 30m
NanoLuciferase Reading
- Prepare a mix of 1/100 dilution of Furimazine in 1X homemade NanoGlow buffer
- Remove cellular supernatant from the 96-well culture plate
- Add 50 µL of diluted substrate to each well
- Measure activity on CentroXS Berthold for 00:00:02 per well
2s