酵母转化方案

This protocol is generalized and not to be considered optimal for all strains, conditions, and applications.  Some aspects of this protocol may have to be modified significantly to suit your particular needs.  It works for simple transformation, targeting in yeast, GAP repair, and multi-plasmid transformations for two-hybrid.

Preparation:

1) Streak or patch yeast on YAPD for 24-48hrs.  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 2xYAPD or SC media (15ml snap cap tubes) from the fresh streak and incubate O/N 30 deg, 200rpm.  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:

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

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) Pellet 3000rpm, 10min at RT in RT6000 or similar centrifuge.

5) While spinning yeast, boil salmon sperm DNA 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 1ml ddH₂O and transfer to 1.5ml tube.

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

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

9) Transfer 100 μl of yeast to a separate 1.5ml 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):

240μl of PEG 3500 w/v

36μl of 1.0M LiAc

50μl of 2mg/ml salmon sperm DNA

34μl or your plasmid(s) + water (1μl of bacterial miniprep DNA is plenty)

360μl TOTAL

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

12) Place yeast at 42 deg for 40min.  For best efficiency the optimal shock time needs to be determined empirically and can vary from 10min to 60min.  For simple single selection plasmid transformation 20-30min usually works fine.

13) Pellet at max speed, 30 sec, RT and remove transformation mix.

14) Add 1ml ddH₂O and use a 1ml pipet tip to ҳtir the cells into solution.  If necessary, pipet the yeast up and down very gently.

15) Plate 200μl on appropriate 100mm or 150mm selective plates and grow 3-4 days at 30deg.  Instead_of_1ml_in_step_14_you_can_also_resuspend_the_yeast_in_400μl and plate the entire amount on one 150mm plate if you desire.