HERP: Haploid Engineering and Replacement Protocol for Saccharomyces
Add the following components to a 2‐L Erlenmeyer flask: Mix to dissolve as much as possible (agar and sulfanilamide won't dissolve until heated).
Autoclave for no more than 20 minutes on a liquid cycle.
Once autoclaved, cool to 50° in a water bath, then add the following and mix: ‐5 g thymidine ‐200 mg methotrexate ‐100 mL 50% (v/v) glycerol, sterilized Pour ~20 mL into plastic petri dishes and allow to set.
You have now made YPGly +AF media.
In a 2‐L Erlenmeyer flask, make 1 L of Synthetic Complete agar using your favorite provider's formulation.
Autoclave then cool to 50° in a water bath.
Dissolve 55 mg of FUdR into 1.1 mL of ddH2O and filter sterilize Add 1 mL of FUdR solution to cooled SC agar and mix.
Pour ~20 mL into plastic petri dishes and allow to set.
You have now made SC +FUdR agar.
Design primers with overhangs that target the cassette to your desired locus.
(See the guidelines for details.)
Amplify the HERP cassette of choice using your targeting primers and a high‐fidelity polymerase such as New England Biolab's Phusion system.
If your reaction makes use of DMSO or other harsh chemicals, clean your PCR product with a column before proceeding.
Culture your strain of choice by inoculating 50 mL of YPD media with enough overnight culture of your strain to bring the OD600 to 0.2‐0.25.
Shake at the optimal temperature for your strain or species until the culture's OD600 reaches 0.85‐1.0.
Shake at the optimal temperature for your strain or species until the culture's OD600 reaches 0.85‐1.0.
Harvest the cells by centrifugation in a 50‐mL conical vial at 3000 RPM for 5 minutes.
Remove supernatant, wash with 25 mL water, and spin at 3000 RPM for 5 minutes.
Remove supernatant and suspend cells in 1 mL of water.
Aliquot 100 μL cell suspension to microcentrifuge tubes, spin for 30 seconds at max speed in a microcentrifuge, and remove supernatant.
Add the following reagents to each cell pellet IN ORDER: Suspend cell pellet in transformation mixture and heat shock.
Once heat‐shocking has been completed, spin the reactions for 30 seconds at max speed, remove the supernatant, and suspend the cells in 600 μL of YPD.
Transfer to glass culture tubes and spin in a culture wheel for 3 hours at the strain's or species' optimal temperature.
Spread 200 μL of recovered cells to each of three YPGly +AF plates.
Only one 200 μL volume of control reaction, however, needs to be plated.
Once all the liquid has been absorbed, store agar up at the optimal temperature.
Colonies will appear in 3‐10 days.
Streak colonies out to fresh YPGly +AF plates.
Analyze by amplifying target locus via PCR and/or sequencing across the insertion junction.
Once you have molecularly confirmed the insertion of the HERP cassette, phenotypically confirm its sensitivity to FUdR by spotting ~1,000 cells onto SC +FUdR plates multiple times.
Sensitive strains should exhibit no growth, while insensitive strains will rapidly grow.
Once your HERP insertion is confirmed and you have established FUdR sensitivity, begin by inoculating the strain in 50 mL of 2X YPA100 +4% galactose (see main text) to an OD600 of 0.2‐0.25 and culture at the optimal temperature.
Once an OD600 of 0.85‐1.0 is reached, repeat steps C4 to C6, except replace the HERP cassette PCR product in C6 with your desired replacement PCR product.
Once the heat shock is completed, remove the supernatant and suspend in 600 μL water.
Spread 200 μL onto each of three SC plates.
Incubate at optimal temperature for 24 hours.
After 24 hours, incubate plates at 4° for one hour.
Lightly replicate plates to SC +FUdR plates.
Re‐replicate to fresh FUdR plates no more than once a day to reduce background growth.
Colonies will appear in 2‐5 days, longer if glucose is replaced by glycerol.
