TITLE: High Efficiency Yeast Library Transformation 
AUTHORS: Benedetta Bolognesi

[DESCRIPTION]
This is a protocol to achieve high-efficiency (10^6 transformants/replicate) when transforming yeast cells with mutational libraries.

[GUIDELINES]
Always work in sterile conditions: flame or microbiological hood.

[STEPS]
SECTION: DAY -2
1. Restreak yeast strains to transform from glycerol stock
SECTION: DAY 0 Pre - growth
2. 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)
SECTION: DAY 0 Pre - growth
3. Prepare all media (see Materials) necessaries for transformation and warm YPDA    in the 30 °C  incubator.

SECTION: DAY 1 Transformation and recovery
4. Measure OD of overnight cultures
SECTION: DAY 1 Transformation and recovery
5. Dilute them in 50ml of YPDA to a final OD of 0.3


 

SECTION: DAY 1 Transformation and recovery
6. Incubate for 210 min to 240 min at 30 °C 

SECTION: DAY 1 Transformation and recovery
7. 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. 

SECTION: DAY 1 Transformation and recovery
8. Pre-label Falcon tubes
SECTION: DAY 1 Transformation and recovery
9. Warm up the centrifuge at  30 °C  . Cold centrifuges will cause your cells to die after heatshock and transformation efficiency to drop.
SECTION: DAY 1 Transformation and recovery
10. After 240 min  measure OD


 



SECTION: DAY 1 Transformation and recovery
11. Pour cells in5 0ml falcon tubes and centrifuge   5 min  3000 x g . Remove supernatant by pouring.

SECTION: DAY 1 Transformation and recovery
12. Resuspend pellet in 10ml of H2O, then bring it up to 50ml.
SECTION: DAY 1 Transformation and recovery
13. Centrifuge 5 min  3000 x g . Remove supernatant by pouring.
SECTION: DAY 1 Transformation and recovery
14. Resuspend in 10ml of SORB (see Materials),transfer to a new falcon, complete to 25ml of SORB

SECTION: DAY 1 Transformation and recovery
15. Centrifuge 5 min  3000 x g . Remove supernatant by pouring.
SECTION: DAY 1 Transformation and recovery
16. Resuspend in 1,42 ml of SORB (see Materials),transfer to a 15ml falcon
SECTION: DAY 1 Transformation and recovery
17. Incubate 30 min on a gentle orbital shaker (tape falcons to shaker)
SECTION: DAY 1 Transformation and recovery
18. 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
SECTION: DAY 1 Transformation and recovery
19. Prepare a lablled 50ml falcon / replicate with 6 ml of Plate Mixture (see Materials)
SECTION: DAY 1 Transformation and recovery
20. 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.
SECTION: DAY 1 Transformation and recovery
21. Turn on water bath at 42 °C 

SECTION: DAY 1 Transformation and recovery
22. Incubate cells for 10 min on the orbital shaker
SECTION: DAY 1 Transformation and recovery
23. Add the 1,4 ml of cells to the 6 ml of plate mixture
SECTION: DAY 1 Transformation and recovery
24. Incubate 30 min on a gentle orbital shaker (tape falcons to shaker)
SECTION: DAY 1 Transformation and recovery
25. Add 600ul of sterile DMSO
SECTION: DAY 1 Transformation and recovery
26. 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). 

SECTION: DAY 1 Transformation and recovery
27. Dry well the top of the tubes to avoid contamination and centrifuge tubes at 3000 rpm .Remove supernatant by pouring.

SECTION: DAY 1 Transformation and recovery
28. Additional quick spin of 30 seconds. Remove remaining liquid with a pipette.
SECTION: DAY 1 Transformation and recovery
29. Resuspend cells in 10ml of Recovery Media (see Materials) and bring it up to 60mls in a  250ml flask
SECTION: DAY 1 Transformation and recovery
30. Shake   30 °C  for 1 hour

SECTION: DAY 1 Transformation and recovery
31. Prepare 50 ml of pre-warmed selection media (e.g. -URA) for each replicate + 40mls for washes
SECTION: DAY 1 Transformation and recovery
32. Centrifuge cells 5 minutes 3000 x g 

SECTION: DAY 1 Transformation and recovery
33. Resuspend cells in 10ml selection media for a wash.
SECTION: DAY 1 Transformation and recovery
34. Centrifuge cells 5 minutes 3000 x g 
SECTION: DAY 1 Transformation and recovery
35. Resuspend cells in 10ml selection media and add them to the 50ml of media in the flask.
SECTION: DAY 1 Transformation and recovery
36. Plate 60 ul  (out of the 60ml) on 2 separate selective plates for each replicate with sterile glass beads.
SECTION: DAY 1 Transformation and recovery
37. Measure OD before putting flasks to grow.
SECTION: DAY 3: From saturated to exponential culture
39. 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. 
SECTION: DAY 1 Transformation and recovery
38. Grow cells for 48-60h    30 °C  
SECTION: DAY 3: From saturated to exponential culture
40. Measure OD 1:10

 

SECTION: DAY 3: From saturated to exponential culture
41. 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.