Editing Pichia EXpression (section)

== Chemical Yeast Transformation Protocol  ==

'''Preparation:''' 

1) Streak or patch yeast on YAPD for 24-48 hrs. Can also use yeast from older plates (3 months at 4 deg) if efficiency is not an issue and the strain cooperates. 

2) Inoculate 2 x 3-5ml cultures in 2 x YAPD or SC media (15ml snap cap tubes) from the fresh streak and incubate O/N 30 deg, 200 rpm. SC media may need to be seeded more heavily (or more tubes may be required). 

YAPD can be substituted for 2xYAPD but efficiency will drop some and yeast will take longer to grow. 

'''Transformation:''' 

<br> 

1. Pre-warm 50 ml 2xYAPD to 30 deg in 500 ml flask (at least 10min). Turn on 42 oC water bath (takes 30 min or more). 

<br> 

2. Dilute O/N culture 1:10 in water and take an OD at 600nm. Use 2xYAPD (or YAPD or SC when appropriate. diluted 1:10 in water as a blank. The diluted culture will usually have an OD of between 0.1 and 1.0. 

3. Dilute culture to approximately 0.1 OD in 50 ml pre-warmed 2xYAPD and incubate 3-6hrs until OD reaches 1.0 (or close). Yeast grown rates vary so be prepared to wait. It usually takes 5-6 hours but can take quite a bit longer. 

EXAMPLE: The diluted culture in step 2 has an OD or 0.2 so the culture OD is 2.0. To get an OD of approximately 0.1 for step 3 you would add 2.5ml of culture to your pre-warmed 50ml 2xYAPD (20x dilution). Obviously its not exact because you will now have 52.5ml instead of 50ml but its close enough. If you want it exact you can simply remove 2.5ml 2xYAPD from the flask before you add your 2.5ml culture but its not necessary. 

4. Spin at 3000 rpm, 10 min at RT. 

5. While spinning yeast, boil salmon sperm DNA for 5 min with boiling cap and chill on ice. Use 3-4 times (storing at -20. and get a new tube. Do not boil over and over again. 

6. Resuspend in 1 ml ddH2O and transfer to 1.5 ml tube. 

7. Vortex 1 min or until yeast are in suspension (no clumps). 

8. Pellet at max speed, 30sec, RT and resuspend in 1 ml ddH2O by vortexing as before. 100 ul will be used for each transformation so there are enough yeast for 10 transformations. 

9. Transfer 100 ul of yeast to a separate 1.5 ml tube for each transformation and pellet at max speed, 30sec, RT and remove supernatant. 

10. For each transformation make up the following (master mix or separately): 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 
| 
|-
| PEG 3500 w/v 
| 240 ul
|-
| 1.0 M LiAc 
| 36 ul
|-
| 2 mg/ml salmon sperm DNA
| 50 ul
|-
| 
plasmid(s). + water

1~5 ul of bacterial miniprep DNA

| 34 ul
|}

360 ul TOTAL

11. Add the transformation mix(es. to the yeast pellets from step #9 and vortex 1 min or until yeast are in suspension (no clumps). 

Optional: add 50 μl DMSO to each transformation and mix immediately. 

12. Place yeast at 42 deg for 40min. For best efficiency the optimal shock time needs to be determined empirically and can vary from 10 min to 60 min. For simple single selection plasmid transformation 15-30 min usually works fine. 

<br> 

13. Pellet at max speed, 30 sec, RT and remove transformation mix.<br>Optional steps: 14 ~ 16 

14. Add 500 ul 5 mM CaCl2 and resuspend cells thoroughly by gently pipeting up and down. 

15. Let tubes stand for at least 5 min (but not longer than 15 min!). at room temperature. 

16. Spin down cells at max speed, 30 sec, RT and remove liquid. 

17. Add 100 ul ddH2O and use a 200 ul pipet tip to stir the cells into solution. If necessary, pipet the yeast up and down very gently. 

18. Plate 100 ul on appropriate 10 cm selective plates and grow 2-4 days at 30 oC. 

<br> 

NOTES:&nbsp; Efficiency varies greatly based on strain and transforming DNA. It can vary from 1×10^3 to 1×10^5 (or so) colonies per ug of input DNA (for plasmid DNA). 

<u>'''Media and Solutions:'''</u> 

'''YAPD (double for 2xYAPD)''': 

10g of BactoYeast extract 

20g of BactoPeptone 

20g Dextrose 

100mg adenine hemisulfate 

q.s. to 1000ml with water. 

Autoclave 20min on liquid cycle. 

'''SC Medium''': 

Selective medium will vary based on strain and application. 

'''Salmon sperm:''' 

2mg/ml in TE. Sigma D1626. Make 100ml in a flask using a magnetic stirrer. Takes 2-4 hours to dissolve. Aliquot in 1.5ml tubes (1ml/tube). 

'''PEG 3500 w/v:''' 

Place 50gm of polyethylene glycol, MW 3350 (Sigma) in a 150 ml glass beaker and add 35 ml of ddH20. Stir for 1-2hrs with a magnetic stirring bar until dissolved. q.s. to 100 ml and keep stirring for at least 10min to mix. Filter using a 0.45 um filter and aliquot in 15ml conicals (10ml each) and store at RT. 

'''LiAc:''' 

1.0M Lithium Acetate. No need to pH. 

<br>