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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 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. Return to Protocols Page |
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