Cloning by Co-Transformation and Recombination in Yeast
Required materials
• Linearized vector
• PCR product to be cloned
• Host yeast strain
• 50% PEG solution (MW=3350):
• 1M Lithium Acetate
• 1M Tris, pH 8.0
• 0.5M EDTA, pH 8.0
• boiled & cooled sheared salmon sperm DNA (10mg/ml)
• DMSO
• Yeast media and plates (depending on markers)
Procedure
1. Inoculate 50 ml YEPD with 0.3 ml liquid stock of PJ69-4a (or any other appropriate yeast strain; we use MATa for activation-domain fusions and MATalpha for baits) in a 250 ml Erlenmeyer flask and grow overnight at 30C (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 (3500 rpm, 1 min, RT) and resuspend all this yeast (combine aliquots) in a total volume of 2ml 0.1M LiOAc.
3. Meanwhile make "1X PEG/LiOAc/TE" mix in a 50 ml tube:
16 ml 50% PEG
2ml 1M LiOAc
200ul Tris
40ul EDTA
500 ul boiled & cooled salmon sperm DNA
~200 ng linear vector DNA
2.24 ml DMSO
Add enough water to bring up total volume to 20ml
***Add the DMSO last and mix quickly after adding by shaking hard for 30 sec or so.
4. Add the 2ml yeast mix and mix hard by hand for 1 min or so. Immediately pour into trough and pipet 200 ul into each of 96 wells ( plus 2 controls) of a 96-well dish (e.g. Costar 3596).
5. Now add 3 ul rePCR products (PCR array shaken and spun before use) or 5 ul Yep351 (= positive control) or nothing ( = negative control). Seal with plastic or Aluminum tape and shake up and down THOROUGHLY.
6. Incubate 30 min at 30°C and heat shock at 42°C for 15 minutes. Let cool to room temp.
7. Spin 7 min at 2500 rpm and aspirate out the supernatant. Add 200 ul 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 30C for 1-3 days. Now record colony number and pick colonies.
• Linearized vector
• PCR product to be cloned
• Host yeast strain
• 50% PEG solution (MW=3350):
• 1M Lithium Acetate
• 1M Tris, pH 8.0
• 0.5M EDTA, pH 8.0
• boiled & cooled sheared salmon sperm DNA (10mg/ml)
• DMSO
• Yeast media and plates (depending on markers)
Procedure
1. Inoculate 50 ml YEPD with 0.3 ml liquid stock of PJ69-4a (or any other appropriate yeast strain; we use MATa for activation-domain fusions and MATalpha for baits) in a 250 ml Erlenmeyer flask and grow overnight at 30C (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 (3500 rpm, 1 min, RT) and resuspend all this yeast (combine aliquots) in a total volume of 2ml 0.1M LiOAc.
3. Meanwhile make "1X PEG/LiOAc/TE" mix in a 50 ml tube:
16 ml 50% PEG
2ml 1M LiOAc
200ul Tris
40ul EDTA
500 ul boiled & cooled salmon sperm DNA
~200 ng linear vector DNA
2.24 ml DMSO
Add enough water to bring up total volume to 20ml
***Add the DMSO last and mix quickly after adding by shaking hard for 30 sec or so.
4. Add the 2ml yeast mix and mix hard by hand for 1 min or so. Immediately pour into trough and pipet 200 ul into each of 96 wells ( plus 2 controls) of a 96-well dish (e.g. Costar 3596).
5. Now add 3 ul rePCR products (PCR array shaken and spun before use) or 5 ul Yep351 (= positive control) or nothing ( = negative control). Seal with plastic or Aluminum tape and shake up and down THOROUGHLY.
6. Incubate 30 min at 30°C and heat shock at 42°C for 15 minutes. Let cool to room temp.
7. Spin 7 min at 2500 rpm and aspirate out the supernatant. Add 200 ul 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 30C for 1-3 days. Now record colony number and pick colonies.