Cloning by co-transformation and recombination in yeast

Required materials:

• PCR product to be cloned	• salmon sperm DNA
• Linearized vector	• DMSO
• Host yeast strain	• 0.1 M Lithium Acetate
• Yeast media and plates (depending on markers)

• 96PEG (100 ml):	45.6 g PEG (Sigma P3640)
	make up to 94.8 ml with H2O
	add 6.1 ml 2 M LiOAc
	1.14 ml 1 M Tris pH 7.5
	232 µl 0.5 M EDTA

(the sum of that is > 100 ml but someone set that up and it worked)

Procedure:

1. Inoculate 50 ml YEPD with 0.3 ml liquid stock of PJ69-4a (or any other appropriate yeast strain; we use Mat a for activation-domain fusions and Mat alpha for baits) in a 250 ml Erlenmeyer flask and grow overnight at 30°C (minimum 15 h, max. 24 h).

   
   

2. Spin out cells in 50 ml conical tube (3500 rpm, 3 min, RT), pour off supernatant, add 2 ml 0.1 M LiOAc and transfer resuspended yeast to 2 microfuge tubes. Spin out yeast and resuspend all this yeast in a total volume of 1.8 ml (incl. yeast) LiOAc (0.1 M).

    

3. Meanwhile make "CT110" in a 50 ml tube
   

   20.73 ml 96PEG

   0.58 ml boiled salmon sperm DNA

   8 µl (200 ng) linear vector DNA

   2.62 ml DMSO

1. Add the DMSO last and mix quickly after adding by shaking hard for 30 sec or so.

   
   

2. Add all yeast and mix hard by hand for 1 min or so. Immediately pour into trough and pipet 245 µl into each of 96 wells ( + 2 controls) of a 96-well dish (e.g., Costar 3596).

   
   

3. Now add 3 µl rePCR products (PCR array shaken and spun before use) or 5 µl Yep351 (= positive control) or nothing ( = negative control). Seal with plastic or aluminum tape and shake on vortex 4 min.

   
   

4. Incubate 30 min at 42°C. Go on immediately or leave plate up to 1 h at RT.

   
   

5. Spin 7 min at 2000 rpm and aspirate supernatant with 8 channel wand. Add 220 µl of water to all 96 wells and then resuspend and plate each well’s yeast one at a time onto 35 mm -Leu (or -Trp) plates. Leave growing at 30°C for 1–3 days. Now record colony number and pick colonies.

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