6. Laboratory scale.
7. Automatic pipettes for 1–5 ml and 100–1000 μl.
8. Glass or plastic funnels (Ø 6 cm).
9. Nylon sieve fitting the funnel (80 μm pore, Ø 8 cm).
10. Tweezers.
11. Scalpel.
12. Sterile 15 ml centrifuge tubes.
13. Sterile 3 ml Pasteur pipettes.
14. Sterile petri dishes Ø 90 mm or 2 ml Eppendorf tubes (see
Note 1).
15. 100 ml and 250 ml glass or plastic beakers.
16. 100–1000 μl and 1–5 ml sterile pipette tips.
17. Preplasmolysis solution (PS) (0.5 M mannitol): dissolve
22.77 g mannitol in dH 2 O, adjust volume to 250 ml with
distilled water (dH 2 O) and pH to 5.6–5.8. Sterilize by autoclaving at 0.1 MPa and 121
C for 15 min. Store at room
temperature (RT).
18. Enzyme mixture solution (ES): dissolve 1 g cellulase Onozuka
R10, 0.1 g pectolyase Y-23, 10.93 g mannitol (0.6 M final
conc.), 0.39 g 2-(N-morpholino)ethanesulfonic acid (MES)
(20 mM final conc.), 0.1 g MgCl 2 Â 6H 2 O (5 mM final
conc.) in dH 2 O (see Note 2). Adjust volume to 100 ml with
dH 2 O and pH to 5.6. Sterilize by filtering using 0.22 μm
syringe filter. Store at 4
C. For long-term storage, place at
À20
C.
19. Sucrose (0.5 M)/MES (1 mM) solution: dissolve 42.79 g
sucrose and 0.05 g MES in dH 2 O. Adjust volume to 250 ml
with dH 2 O and pH to 5.8. Sterilize by autoclaving at 0.1 MPa
and 121
C for 15 min. Store at RT.
20. W5 solution: dissolve 2.25 g NaCl (154 mM final conc.),
4.59 g CaCl 2 Â 2H 2 O (125 mM final conc.), 0.09 g KCl
(5 mM final conc.) and 0.23 g glucose (5 mM final conc.) in
dH 2 O. Adjust volume to 250 ml with dH 2 O and pH to 5.8.
Sterilize by autoclaving at 0.1 MPa and 121
C for 15 min.
Store at RT.
2.1.3 Extraction
of Carotenoid Crystals
1. Centrifuge with cooling to 4
C capable to achieve 26,000 Â g
and equipped with a rotor for 2 ml tubes.
2. Automatic 1000 μl pipette.
3. 1000 μl pipette tips.
4. 1.5 ml Eppendorf tubes.
5. 2 ml Eppendorf tubes with round shaped bottom.
248
Tomasz Oleszkiewicz et al.
7. Automatic pipettes for 1–5 ml and 100–1000 μl.
8. Glass or plastic funnels (Ø 6 cm).
9. Nylon sieve fitting the funnel (80 μm pore, Ø 8 cm).
10. Tweezers.
11. Scalpel.
12. Sterile 15 ml centrifuge tubes.
13. Sterile 3 ml Pasteur pipettes.
14. Sterile petri dishes Ø 90 mm or 2 ml Eppendorf tubes (see
Note 1).
15. 100 ml and 250 ml glass or plastic beakers.
16. 100–1000 μl and 1–5 ml sterile pipette tips.
17. Preplasmolysis solution (PS) (0.5 M mannitol): dissolve
22.77 g mannitol in dH 2 O, adjust volume to 250 ml with
distilled water (dH 2 O) and pH to 5.6–5.8. Sterilize by autoclaving at 0.1 MPa and 121
C for 15 min. Store at room
temperature (RT).
18. Enzyme mixture solution (ES): dissolve 1 g cellulase Onozuka
R10, 0.1 g pectolyase Y-23, 10.93 g mannitol (0.6 M final
conc.), 0.39 g 2-(N-morpholino)ethanesulfonic acid (MES)
(20 mM final conc.), 0.1 g MgCl 2 Â 6H 2 O (5 mM final
conc.) in dH 2 O (see Note 2). Adjust volume to 100 ml with
dH 2 O and pH to 5.6. Sterilize by filtering using 0.22 μm
syringe filter. Store at 4
C. For long-term storage, place at
À20
C.
19. Sucrose (0.5 M)/MES (1 mM) solution: dissolve 42.79 g
sucrose and 0.05 g MES in dH 2 O. Adjust volume to 250 ml
with dH 2 O and pH to 5.8. Sterilize by autoclaving at 0.1 MPa
and 121
C for 15 min. Store at RT.
20. W5 solution: dissolve 2.25 g NaCl (154 mM final conc.),
4.59 g CaCl 2 Â 2H 2 O (125 mM final conc.), 0.09 g KCl
(5 mM final conc.) and 0.23 g glucose (5 mM final conc.) in
dH 2 O. Adjust volume to 250 ml with dH 2 O and pH to 5.8.
Sterilize by autoclaving at 0.1 MPa and 121
C for 15 min.
Store at RT.
2.1.3 Extraction
of Carotenoid Crystals
1. Centrifuge with cooling to 4
C capable to achieve 26,000 Â g
and equipped with a rotor for 2 ml tubes.
2. Automatic 1000 μl pipette.
3. 1000 μl pipette tips.
4. 1.5 ml Eppendorf tubes.
5. 2 ml Eppendorf tubes with round shaped bottom.
248
Tomasz Oleszkiewicz et al.