Silver: Time-Lapse Microscopy

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Pad Preparation

1. Microwave 2% agarose (mix of low-melt and normal, to taste) in Thorn media (see below). (If you have used different percentages of agarose pads please let us know, bruno)

2. Apply 2mL to a 24x60 mm coverslip.

3. Cover with additional coverslip, creating a sandwich. Let cool for 30 minutes. (1h seems to work better)

Cell Preparation

4. Grow 5mL culture in Thorn media to OD 600 = 0.3. Back dilute if necessary.

5. Centrifuge at 2000 rpm for 2 minutes. Resuspend in 1mL Thorn media and transfer to a 1.5mL eppendorf tube.

6. Spin in a microfuge for 15 seconds to pellet. Resuspend in 100-200 µL Thorn media.

7. Once cooled cut a 5x5 mm square from the agarose pad. Apply 0.2 µL of cells to the top surface.

8. Invert square onto a glass-bottom imaging dish. Add a few drops of water to the dish (for moisture), and cover edges with parafilm.

9. Image.

Thorn Media

Standard media except with low-fluorescence yeast nitrogen base.

(Yeast nitrogen base without riboflavin and folic acid.)

• 5 g/l (NH4)₂SO₄
• 1 g/l KH₂PO₄
• 0.5 g/l MgSO₄
• 0.1 g/l NaCl
• 0.1 g/l Ca₂Cl
• 0.5 mg/l H₃BO₄
• 0.04 mg/l CuSO₄
• 0.1 mg/l KI
• 0.2 mg/l FeCl₃
• 0.4 mg/l MnSO₄
• 0.2 mg/l Na₂MoO₄
• 0.4 mg/l ZnSO₄
• 2 µg/l biotin
• 0.4 mg/l calcium pantothenate
• 2 mg/l inositol
• 0.4 mg/l niacin
• 0.2 mg/l PABA
• 0.4 mg/l pyridoxine HCl
• 0.4 mg/l thiamine

(Sheff MA, Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 2004; 21: 661–670.)