Editing Pichia EXpression (section)

== Yeast glycerol stock strain preparation  ==

<br> PichiaPink strains: PPS1, S2, S3 and S4 

Streak each strain on a YPDa agar plate and grow 3 days at 29C to obtain single colonies (most are white color colonies). All 4 strains are grown well after 3 days. 

Preparation of stock strains:<br>'''Day 1'''<br>Starter culture: Culture two of a single colony of each strain in 10 ml YPDa medium in a 50-ml conical tube with cap (total 2 x 4) for 16-24 hours (starting at 10am) at 29C and shaking at 250 rpm. <br>(note: it is important to have the media to flask volume ratio: at least 1:5) 

'''Day 2 '''<br>Seed 200 ml YPDa with the starter culture to an OD600 of 0.2. Grow at shaking for 1-2days at 29C to an OD600 of 2-3. 

'''Day 3 '''<br>-- Harvest cells at 1500 x g for 5 min at 4C using 200-ml Beckaman coulter bottle (autoclaved). Alternatively, harvest cells by dividing 200 ml into 4 x 50-ml conical tube of pathogen free.<br>-- Resuspend the cells into YPDa containing 25% glycerol by adding to a final OD600 of 50-100. For 200 ml cell pellet, 4 ml of the YPD media is added and measure its OD at 600nm to an OD600nm of 90 as measured. 

'''Media''' used for stock strain preparation<br>'''YPD''': 1% yeast extract and 2% peptone (YP autoclaved) and 2% of dextrose (by adding 20% sterilized dextrose to the autoclaved YP medium 

'''YPDa''': Add 0.2% sterilized adenine (as 75X) to the YPD before culture (dilute 75x) in the YPD. 

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'''Invitrogen Protocol'''<br> 

To store cells for weeks to months, use YPD medium and YPD agar slants<br> 

<br>1. Streak each strain to obtain single colonies on YPD agar plates. Grow 3–5 days at 24–30°C.<br>2. Transfer one colony to a YPD stab and grow for 3–5 days at 30°C.<br>3. You can store the cells on YPD for several weeks at 4°C.<br>To store cells for months to years, store frozen at –80°C.<br>'''Day 1''':<br>1. Culture a single colony of each strain in 10 mL of YPD medium for 16–20 hours at 24–30°C, shaking at 300 rpm. This is your starter culture.<br>Note: It is important to have adequate aeration for growth. Always use 1:5 ratio of media to flask volume.<br>'''Day 2''':<br>2. Seed 200 mL of YPD medium with the starter culture to an OD600 of 0.2. Grow shaking for 1–2 days at 24–30°C to an OD600 of 2–3.<br>'''Day 3 or 4''':<br>3. Harvest the cells by centrifuging at 1,500 × g for 5 minutes. Remove the supernatant and resuspend the cells in YPD medium containing 25% glycerol to<br> a final OD600 of 50–100 (approximately 2.5–5.0 × 10<sup>9</sup> cells/mL).<br>4. Aliquot the cells in cryovials (1 mL aliquots) and freeze in liquid nitrogen or a dry ice/ethanol bath and store at –80°C. Cells will be pink in color.<br><br>