Materials and Reagents

1. Consumables

   1. Test tubes (Fisherbrand, catalog number: 14-961-34, or equivalent)

   2. Standard sterile serological pipettes (2, 5, 10, and 25 ml)

   3. Micropipette tips (1,000-100 µl, 200-20 µl, 20-2 µl, and 2.0-0.1 µl)

   4. 100 × 15 mm Petri dishes (VWR, catalog number: 25384-088 or equivalent)

   5. 15 and 50 ml centrifuge tubes (Corning, catalog numbers: 430766 and 430291, respectively, or equivalent)




2. Medium and growth conditions

   Standard pombe minimal glutamate (PMG) medium, pH 5.6, supplemented as necessary with 250 mg/L each of adenine, uracil, leucine, histidine, and lysine (Sabatinos and Forsburg, 2010). Bacto agar should be added at a final concentration of 2% (w/v) for preparation of solid agar medium. Leu-PMG medium has all supplements mentioned except leucine. Add thiamine as specified in the methods. Thiamine must be added after cooling the media to 45-50°C after autoclaving for preparation of PMG agar medium with thiamine.

   
   

   The base pTIN-system vectors, pTIN and pTIN7m, are available at: https://www.addgene.org/Stewart_Shuman.

   pTIN   ID#162885

   pTIN7m    ID#162886

   Your GOI can be cloned into and overexpressed from these vectors.

   
   

   1. Adenine (Sigma, catalog number: A2786)

   2. Uracil (catalog number: U0750)

   3. Leucine (Fisher, catalog number: BP385-100)

   4. Histidine (Sigma, catalog number: H5659)

   5. Lysine (Fisher, catalog number: BP386-100)

   6. Bacto agar (BD, catalog number: 214010)

   7. Thiamine hydrochloride (Sigma, catalog number: T1270)

   8. Bacto agar (BD, catalog number: 214010)




3. Yeast competent cells and transformation

   1. Tris base (Fisher, catalog number: BP152)

   2. EDTA (Sigma, catalog number: E5134)

   3. Lithium acetate (LiOAc) (Sigma, catalog number: L4158)

   4. PEG 3350 (Sigma, catalog number: P4338)

   5. Glycerol (Fisher, catalog number: BP229)

   6. Salmon sperm DNA (ssDNA) (Sigma, catalog number: D1626)

   7. LiOAc-mix (see Recipes)

   8. PEG-mix (see Recipes)

   9. 50% Glycerol/LiOAc-mix (see Recipes)

   10. Salmon sperm DNA (ssDNA) solution (see Recipes)

   11. 2,000× Thiamine hydrochloride (see Recipes)

Equipment

1. Incubator shaker (New Brunswick Innova 44 or equivalent)

2. Micropipettes (1,000-100 µl, 200-20 µl, 20-2 µl, and 2.0-0.1 µl)

3. Standard glass Erlenmeyer flasks (volume of culture should not exceed half the volume of the flask)

4. Incubators (Nuaire IR autoflow or equivalent)

5. Test tube roller (New Brunswick, TC-7 with 25 mm test tube holder or equivalent)

6. Microcentrifuge (Eppendorf 5425 or equivalent)

7. Microcentrifuge tubes (Crystalgen, catalog number: L-2052 or equivalent)

8. Glass beads (Fisher Scientific, catalog number: 11-312C or equivalent)

9. Cooling centrifuge (Thermo Scientific Sorvall BP8 with HAEMAFlex 6 swinging bucket rotor or equivalent)

10. Centrifuge bottles (Thermo Scientific, catalog number: 3120-0250 or equivalent)

Procedure