High Efficiency Yeast Library Transformation

Restreak yeast strains to transform from glycerol stock

For each biological replicate, put 1 individual yeast colony to grow in 3 mL YPDA in culture tubes (choose three colonies that are similar in size and not too big, not too little)

Prepare all media (see Materials) necessaries for transformation and warm YPDA in the 30°C incubator.

Measure OD of overnight cultures

Dilute them in 50ml of YPDA to a final OD of 0.3

A	B	C	D

Incubate for 3h 30m 0s to 4h 0m 0s at 30°C

Prewarm reagents 30°C Water, SORB, Plate mixture, Recovery Media and Plasmid selection media. You can also prepare the flasks with the corresponding media while the cells grow and warm the flasks directly.

Pre-label Falcon tubes

Warm up the centrifuge at 30°C . Cold centrifuges will cause your cells to die after heatshock and transformation efficiency to drop.

After 4h 0m 0s measure OD

A

Pour cells in5 0ml falcon tubes and centrifuge 5 min 3000x g,0h 0m 0s . Remove supernatant by pouring.

Resuspend pellet in 10ml of H2O, then bring it up to 50ml.

Centrifuge 5 min 3000x g,0h 0m 0s . Remove supernatant by pouring.

Resuspend in 10ml of SORB (see Materials),transfer to a new falcon, complete to 25ml of SORB

Centrifuge 5 min 3000x g,0h 0m 0s . Remove supernatant by pouring.

Resuspend in 1,42 ml of SORB (see Materials),transfer to a 15ml falcon

Incubate 30 min on a gentle orbital shaker (tape falcons to shaker)

In the meantime, boil 45 ul (per replicate) of 10mg/ml ssDNAfor 5 min and let it sit on ice for at least 2 min

Prepare a lablled 50ml falcon / replicate with 6 ml of Plate Mixture (see Materials)

After 30 min on the orbital shaker, add 1ug of plasmid library and 45ul of ssDNA and let cells sit at RT for 5 min.

Turn on water bath at 42°C

Incubate cells for 10 min on the orbital shaker

Add the 1,4 ml of cells to the 6 ml of plate mixture

Incubate 30 min on a gentle orbital shaker (tape falcons to shaker)

Add 600ul of sterile DMSO

Heat shock for 20 min at 42°C in the water bath. Quickly take out tubes and and shake them upside down at 30’’, 1’, 1’30’’, 2’, 2’30’’, 5’, 10’ and 15’. Falcons 50ml must be mostly submerged but water should not reach the tube-limit level as this may cause contamination. Bring a timer with you and some weight to keep the tube-rack down and avoid floating (another rack or something heavier).

Dry well the top of the tubes to avoid contamination and centrifuge tubes at 3000rpm,0h 0m 0s .Remove supernatant by pouring.

Additional quick spin of 30 seconds. Remove remaining liquid with a pipette.

Resuspend cells in 10ml of Recovery Media (see Materials) and bring it up to 60mls in a 250ml flask

Shake 200rpm 30°C for 1 hour

Prepare 50 ml of pre-warmed selection media (e.g. -URA) for each replicate + 40mls for washes

Centrifuge cells 5 minutes 3000x g,0h 0m 0s

Resuspend cells in 10ml selection media for a wash.

Centrifuge cells 5 minutes 3000x g,0h 0m 0s

Resuspend cells in 10ml selection media and add them to the 50ml of media in the flask.

Plate 60 ul (out of the 60ml) on 2 separate selective plates for each replicate with sterile glass beads.

Measure OD before putting flasks to grow.

Grow cells for 48-60h 200rpm 30°C

Note: This step might introduce bottlenecking. It’s a healthy practice to calculate how many cells from the saturated culture you are using to then grow the exponential culture, and if they cover your library well. Double check that the volume of cells you pipette will have enough cells to cover 100x (or more) each variant. Consider that 50% of the cells might be dead due to the saturation of the culture.

Measure OD 1:10

A	B

Pipette the calculated volume in flasks with 60ml pre-warmed selection media. Keep track of time. This culture with grow to exponential before protein expression/selection/competition are carried out.