12. Centrifuge Allegra™ X-22R equipped with a rotor for 50-ml
tubes or equivalent.
2.4 qPCR Expression
Analyses
1. 0.5 mm zirconia/silica beads.
2. Screw-cap microtubes of 2 ml, without skirted base.
3. RNeasy Plant Mini Kit (QIAGEN).
4. β-mercaptoethanol.
5. DNase I recombinant, RNase-free.
6. Transcriptor First Strand cDNA synthesis kit (Roche).
7. Primers:
RtFfcarS-1F: 5
0 -GATACCCGGCGGAAAGGTTA-3
0 .
RtFfcarS-1R: 5
0 -CTGACAGTCCATTTCAGCGC-3
0 .
tub-1F: 5
0 -CCGGTGCTGGAAACAACTG-3
0 .
tub-1R: 5
0 -CGAGGACCTGGTCGACAAGT-3
0 .
8. LightCycler
® 480 SYBR Green I Master 5 Â 1 ml (Roche).
9. LightCycler
® 480 Instrument (Roche Life Science).
10. Microcentrifuge with a rotor for microcentrifuge tubes
(1.5–2 ml) at 12,000 Â g.
2.5 Carotenoids
Extraction
1. Washed sea sand.
2. Screw-cap microtubes of 2 ml without skirted base.
3. Acetone.
4. Lyophilizer.
5. FAST-PREP24 (Biomedicals).
6. Speed vacuum concentrator.
7. UV/visible scanning spectrophotometer.
8. UV cuvette (1-ml volume) and 8-mm high micro cell cuvette
(0.1-ml).
2.6 General
Equipment
1. Platform shaker.
2. Thermocycler.
3. Vortex.
4. Standard fume hood.
3 Methods
3.1 Construction of a
Plasmid with a
Carotenoid Regulator
Controlled by Tet-on
System
1. Perform a DNA extraction of plasmid pLR14 that contains the
gene of interest, carS [12].
2. Add PmeI-restriction sites to carS ORF by PCR amplification
using primers carS-PmeI-1F and carS-PmeI-1R.
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Julia Marente et al.
tubes or equivalent.
2.4 qPCR Expression
Analyses
1. 0.5 mm zirconia/silica beads.
2. Screw-cap microtubes of 2 ml, without skirted base.
3. RNeasy Plant Mini Kit (QIAGEN).
4. β-mercaptoethanol.
5. DNase I recombinant, RNase-free.
6. Transcriptor First Strand cDNA synthesis kit (Roche).
7. Primers:
RtFfcarS-1F: 5
0 -GATACCCGGCGGAAAGGTTA-3
0 .
RtFfcarS-1R: 5
0 -CTGACAGTCCATTTCAGCGC-3
0 .
tub-1F: 5
0 -CCGGTGCTGGAAACAACTG-3
0 .
tub-1R: 5
0 -CGAGGACCTGGTCGACAAGT-3
0 .
8. LightCycler
® 480 SYBR Green I Master 5 Â 1 ml (Roche).
9. LightCycler
® 480 Instrument (Roche Life Science).
10. Microcentrifuge with a rotor for microcentrifuge tubes
(1.5–2 ml) at 12,000 Â g.
2.5 Carotenoids
Extraction
1. Washed sea sand.
2. Screw-cap microtubes of 2 ml without skirted base.
3. Acetone.
4. Lyophilizer.
5. FAST-PREP24 (Biomedicals).
6. Speed vacuum concentrator.
7. UV/visible scanning spectrophotometer.
8. UV cuvette (1-ml volume) and 8-mm high micro cell cuvette
(0.1-ml).
2.6 General
Equipment
1. Platform shaker.
2. Thermocycler.
3. Vortex.
4. Standard fume hood.
3 Methods
3.1 Construction of a
Plasmid with a
Carotenoid Regulator
Controlled by Tet-on
System
1. Perform a DNA extraction of plasmid pLR14 that contains the
gene of interest, carS [12].
2. Add PmeI-restriction sites to carS ORF by PCR amplification
using primers carS-PmeI-1F and carS-PmeI-1R.
348
Julia Marente et al.