3. 24 Â 60 mm Cover glass.
4. Physiological salt solution: 0.9% NaCl.
5. 30
C Incubator.
3 Methods
3.1 Construction of
Csy4∗ Expression
Plasmids for Imaging
in Plants
1. Recombine the attB1-Csy4∗-KpnI-NLS-KpnI-attB2 gene
fragment with pDONR221 using Gateway BP Clonase II
according to the manufacturer’s instructions.
2. Electroporate 1 μL BP recombination reaction into 50 μL XL1
Blue MRF’ cells, and then incubate cells in 1 mL LB medium
without antibiotics at 37
C for 1 h.
3. Plate 100 μL transformed cells on LB-agar plates containing
50 μg/mL kanamycin, and incubate overnight at 37
C.
4. The next day, inoculate several well-separated colonies into
5 mL LB liquid medium containing 50 μg/mL kanamycin
each, and incubate overnight at 37
C with shaking.
5. Purify pDONR221.Csy4∗-KpnI-NLS-KpnI plasmid DNA
from each culture using a plasmid miniprep kit according to
the manufacturer’s instructions.
6. Verify the correct insert by DNA sequencing with primers
DONRfor and DONRrev.
7. With the miniprepped DNA of one correct clone, set up the
following restriction digest according to the manufacturer’s
instructions and incubate at 37
C for 2 h: 10 μL
pDONR221.Csy4∗-KpnI-NLS-KpnI, 5 μL 10Â buffer, and
1 μL KpnI, and make up to 50 μL with water.
8. Purify the DNA from the restriction digests with a PCR
cleanup kit according to the manufacturer’s instructions. A
size cutoff of greater than 50 bp will ensure that the excised
NLS fragment passes through the column.
9. Re-ligate 2 μL of the purified digested plasmid in a total volume
of 20 μL using T4 DNA ligase according to the manufacturer’s
instructions.
10. Electroporate 2 μL ligation reaction into 50 μL XL1 Blue
MRF’ cells, and then incubate cells in 1 mL LB medium
without antibiotics at 37
C for 1 h (see Note 5).
11. Plate 25 μL transformed cells on LB-agar plates containing
50 μg/mL kanamycin, and incubate overnight at 37
C.
12. The next day, inoculate several 5 mL aliquots of LB liquid
medium containing 50 μg/mL kanamycin with a wellseparated colony each, and incubate overnight at 37
C with
shaking.
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David Burnett et al.
4. Physiological salt solution: 0.9% NaCl.
5. 30
C Incubator.
3 Methods
3.1 Construction of
Csy4∗ Expression
Plasmids for Imaging
in Plants
1. Recombine the attB1-Csy4∗-KpnI-NLS-KpnI-attB2 gene
fragment with pDONR221 using Gateway BP Clonase II
according to the manufacturer’s instructions.
2. Electroporate 1 μL BP recombination reaction into 50 μL XL1
Blue MRF’ cells, and then incubate cells in 1 mL LB medium
without antibiotics at 37
C for 1 h.
3. Plate 100 μL transformed cells on LB-agar plates containing
50 μg/mL kanamycin, and incubate overnight at 37
C.
4. The next day, inoculate several well-separated colonies into
5 mL LB liquid medium containing 50 μg/mL kanamycin
each, and incubate overnight at 37
C with shaking.
5. Purify pDONR221.Csy4∗-KpnI-NLS-KpnI plasmid DNA
from each culture using a plasmid miniprep kit according to
the manufacturer’s instructions.
6. Verify the correct insert by DNA sequencing with primers
DONRfor and DONRrev.
7. With the miniprepped DNA of one correct clone, set up the
following restriction digest according to the manufacturer’s
instructions and incubate at 37
C for 2 h: 10 μL
pDONR221.Csy4∗-KpnI-NLS-KpnI, 5 μL 10Â buffer, and
1 μL KpnI, and make up to 50 μL with water.
8. Purify the DNA from the restriction digests with a PCR
cleanup kit according to the manufacturer’s instructions. A
size cutoff of greater than 50 bp will ensure that the excised
NLS fragment passes through the column.
9. Re-ligate 2 μL of the purified digested plasmid in a total volume
of 20 μL using T4 DNA ligase according to the manufacturer’s
instructions.
10. Electroporate 2 μL ligation reaction into 50 μL XL1 Blue
MRF’ cells, and then incubate cells in 1 mL LB medium
without antibiotics at 37
C for 1 h (see Note 5).
11. Plate 25 μL transformed cells on LB-agar plates containing
50 μg/mL kanamycin, and incubate overnight at 37
C.
12. The next day, inoculate several 5 mL aliquots of LB liquid
medium containing 50 μg/mL kanamycin with a wellseparated colony each, and incubate overnight at 37
C with
shaking.
166
David Burnett et al.