5. Add 5 μL of 10 mg/mL ssDNA denatured at 95
C for 10 min.
6. Add 500 ng–1 μg of plasmid expressing MCP-GFP (i.e.,
YcpLac111 MCP-NLS-2xyeGFP) for each transformation.
7. Add 150 μL of cells to the tube and mix by gentle vortexing
(speed 5 out of 10).
8. Incubate at room temperature for 30 min.
9. Heat shock the cells at 42
C for 15 min.
10. Centrifuge the cells for 3 min at 7000 Â g.
11. Discard the supernatant, resuspend the pellet in 100 μL of
DDW, and plate the entire transformation on selective LEU
plates.
12. Incubate at 26
C for 3–4 days. The transformed cells can be
used to start cultures for live imaging (see Note 8).
3.1.2 Growing Yeast
Cells for Live Imaging
1. Grow a low-density culture of the yeast strain expressing the
endogenously tagged mRNA and MCP-GFP in selective LEU
medium overnight at 26
C. Apply constant shaking at
210 rpm. It is important to keep the cells growing in exponential phase (OD 600 <1) at all times.
2. In the morning, dilute the cells with fresh medium to an OD 600
~0.1 and allow to grow until OD 600 0.2–0.3. At this concentration, the autofluorescence of the cells is minimal and the
expression of the MCP is homogenous (see Note 9).
3.1.3 Coating of Delta-T
Dishes and Plating of Cells
1. Incubate the Delta-T dishes with 400 μL of ConA at a final
concentration 1 mg/mL for 10 min at room temperature.
2. Aspirate the excess and let the dish air-dry completely.
3. Activate the ConA coating, by incubating the dish with 400 μL
of ConA activating solution, for 10 min at room temperature
(see Note 10).
4. Aspirate the excess and let the dish air-dry completely.
5. Wash the dish twice with sterile DDW and let air-dry
completely.
6. Plate 500 μL of cells at OD 600 0.2–0.3 (see Note 11).
7. Place the dish on the microscope stage and let the cells attach
for at least 15–30 min. Using the Delta-T temperature control
system, allow the temperature to stabilize at 26
C. It is important to wait until cells attach and restart the cell cycle.
3.1.4 Live Imaging
Acquisition
The live imaging conditions need to be adapted based on the
expression of the mRNA under investigation. We recommend testing the mRNA of interest first by smFISH. Here, we outline the
imaging conditions for the constitutive MDN1 mRNA. We previously characterized the expression of this mRNA by smFISH
Imaging Single mRNAs in Living Eukaryotic Cells
133
C for 10 min.
6. Add 500 ng–1 μg of plasmid expressing MCP-GFP (i.e.,
YcpLac111 MCP-NLS-2xyeGFP) for each transformation.
7. Add 150 μL of cells to the tube and mix by gentle vortexing
(speed 5 out of 10).
8. Incubate at room temperature for 30 min.
9. Heat shock the cells at 42
C for 15 min.
10. Centrifuge the cells for 3 min at 7000 Â g.
11. Discard the supernatant, resuspend the pellet in 100 μL of
DDW, and plate the entire transformation on selective LEU
plates.
12. Incubate at 26
C for 3–4 days. The transformed cells can be
used to start cultures for live imaging (see Note 8).
3.1.2 Growing Yeast
Cells for Live Imaging
1. Grow a low-density culture of the yeast strain expressing the
endogenously tagged mRNA and MCP-GFP in selective LEU
medium overnight at 26
C. Apply constant shaking at
210 rpm. It is important to keep the cells growing in exponential phase (OD 600 <1) at all times.
2. In the morning, dilute the cells with fresh medium to an OD 600
~0.1 and allow to grow until OD 600 0.2–0.3. At this concentration, the autofluorescence of the cells is minimal and the
expression of the MCP is homogenous (see Note 9).
3.1.3 Coating of Delta-T
Dishes and Plating of Cells
1. Incubate the Delta-T dishes with 400 μL of ConA at a final
concentration 1 mg/mL for 10 min at room temperature.
2. Aspirate the excess and let the dish air-dry completely.
3. Activate the ConA coating, by incubating the dish with 400 μL
of ConA activating solution, for 10 min at room temperature
(see Note 10).
4. Aspirate the excess and let the dish air-dry completely.
5. Wash the dish twice with sterile DDW and let air-dry
completely.
6. Plate 500 μL of cells at OD 600 0.2–0.3 (see Note 11).
7. Place the dish on the microscope stage and let the cells attach
for at least 15–30 min. Using the Delta-T temperature control
system, allow the temperature to stabilize at 26
C. It is important to wait until cells attach and restart the cell cycle.
3.1.4 Live Imaging
Acquisition
The live imaging conditions need to be adapted based on the
expression of the mRNA under investigation. We recommend testing the mRNA of interest first by smFISH. Here, we outline the
imaging conditions for the constitutive MDN1 mRNA. We previously characterized the expression of this mRNA by smFISH
Imaging Single mRNAs in Living Eukaryotic Cells
133