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 .
