select a cell line with optimal MCP-FP expression (Subheading
3.2.3), how to prepare the cells (Subheading 3.2.4), and how to
perform live image acquisition (Subheading 3.2.5).
2 Materials
2.1 Materials
for Visualizing
MBSV6-Labeled
mRNAs in Yeast
1. Yeast cells: All strains used in this protocol are derived from the
S. cerevisiae background BY4741 MATa; his3Δ1; leu2Δ0;
met15Δ0; ura3Δ0.
2. YPD medium: 50 g/L of the YPD mix (i.e., Clonetech). Sterilize by autoclaving.
3. LEU medium (dropout media lacking leucine): 6.7 g/L Yeast
nitrogen base (YNB) with ammonium sulfate, dropout mix
lacking leucine, 20 g/L glucose. Sterilize by autoclaving.
4. LEU plates (dropout agar plates): 6.7 g/L Yeast nitrogen base
(YNB) with ammonium sulfate, dropout mix lacking leucine,
20 g/L glucose, 20 g/L of bacteriological agar. Sterilize by
autoclaving.
5. 100% Glycerol stock: Sterilize by autoclaving. Store at room
temperature, protected from light.
6. Plasmids: pET264-pUC 24xMS2V6 Loxp KANr Loxp
(Addgene ID:104393), pET251-pUC 12xMS2V6 Loxp
KANr Loxp (Addgene ID:104392); pET296-YcpLac111
CYC1p-MCP-NLS-2xyeGFP
(yeast-optimized
eGFP)
(Addgene ID:104394); pET511-YcpLac111 CYC1p-MCPNLS-3xyeGFP; pET518-YcpLac11-CYC1p-MCP-1x-eGFP;
pET519-YcpLac11-CYC1p-MCP-1xEnvy;
pET521YcpLac11-CYC1p-MCP-1xmuGFP;
pET522-YcpLac11CYC1p-MCP-1xmNG;
pET523-YcpLac11-CYC1p-MCP1xsfGFP (constructs available upon request).
7. Lithium-TE: 100 mM LiAc, 10 mM Tris–HCl, pH 7.5, 1 mM
EDTA. Sterilize by autoclaving.
8. Lithium-TE-PEG: 100 mM LiAc, 10 mM Tris, pH 7.5, 1 mM
EDTA, 50% PEG 3350-4000. Sterilize by autoclaving.
9. Salmon sperm DNA (ssDNA): 10 mg/mL Lyophilized,
sheared, organically extracted, and denatured ssDNA is resuspended in double-distilled water (DDW). Store 100 μL aliquots at À20
C.
10. Centrifuges (table top): Up to 20,000 Â g for samples 1.5 mL.
11. Heat blocks at 42
C and 95
C.
12. Temperature-controlled shaker for yeast cultures.
13. Temperature-controlled, Delta-T dishes (i.e., Bioptech Cat#
04200417C).
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Xavier Pichon et al.
3.2.3), how to prepare the cells (Subheading 3.2.4), and how to
perform live image acquisition (Subheading 3.2.5).
2 Materials
2.1 Materials
for Visualizing
MBSV6-Labeled
mRNAs in Yeast
1. Yeast cells: All strains used in this protocol are derived from the
S. cerevisiae background BY4741 MATa; his3Δ1; leu2Δ0;
met15Δ0; ura3Δ0.
2. YPD medium: 50 g/L of the YPD mix (i.e., Clonetech). Sterilize by autoclaving.
3. LEU medium (dropout media lacking leucine): 6.7 g/L Yeast
nitrogen base (YNB) with ammonium sulfate, dropout mix
lacking leucine, 20 g/L glucose. Sterilize by autoclaving.
4. LEU plates (dropout agar plates): 6.7 g/L Yeast nitrogen base
(YNB) with ammonium sulfate, dropout mix lacking leucine,
20 g/L glucose, 20 g/L of bacteriological agar. Sterilize by
autoclaving.
5. 100% Glycerol stock: Sterilize by autoclaving. Store at room
temperature, protected from light.
6. Plasmids: pET264-pUC 24xMS2V6 Loxp KANr Loxp
(Addgene ID:104393), pET251-pUC 12xMS2V6 Loxp
KANr Loxp (Addgene ID:104392); pET296-YcpLac111
CYC1p-MCP-NLS-2xyeGFP
(yeast-optimized
eGFP)
(Addgene ID:104394); pET511-YcpLac111 CYC1p-MCPNLS-3xyeGFP; pET518-YcpLac11-CYC1p-MCP-1x-eGFP;
pET519-YcpLac11-CYC1p-MCP-1xEnvy;
pET521YcpLac11-CYC1p-MCP-1xmuGFP;
pET522-YcpLac11CYC1p-MCP-1xmNG;
pET523-YcpLac11-CYC1p-MCP1xsfGFP (constructs available upon request).
7. Lithium-TE: 100 mM LiAc, 10 mM Tris–HCl, pH 7.5, 1 mM
EDTA. Sterilize by autoclaving.
8. Lithium-TE-PEG: 100 mM LiAc, 10 mM Tris, pH 7.5, 1 mM
EDTA, 50% PEG 3350-4000. Sterilize by autoclaving.
9. Salmon sperm DNA (ssDNA): 10 mg/mL Lyophilized,
sheared, organically extracted, and denatured ssDNA is resuspended in double-distilled water (DDW). Store 100 μL aliquots at À20
C.
10. Centrifuges (table top): Up to 20,000 Â g for samples 1.5 mL.
11. Heat blocks at 42
C and 95
C.
12. Temperature-controlled shaker for yeast cultures.
13. Temperature-controlled, Delta-T dishes (i.e., Bioptech Cat#
04200417C).
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