3. Freshly prepared floral dip transformation solution (5%
sucrose, 0.03% Silwet L-77).
4. Antibiotics: kanamycin, hygromycin, carbenicillin, and
rifampicin.
5. Centrifuge, 500 mL centrifuge tubes.
6. 5% Sodium hypochlorite solution.
7. 0.05% Agar (autoclaved).
8. Transgenic plant selection solid medium: 0.5Â Murashige and
Skoog (MS) with vitamins (Phyto Technology Laboratories,
#M524), 0.8% agar, 30 mg/L hygromycin B, 50 mg/L
carbenicillin.
9. Plant soils, pots, and trays.
10. Growth chambers and greenhouse.
2.4 Screening of
Mutations or Base
Editings
1. CTAB buffer: 2% cetyltrimethylammonium bromide (CTAB),
1.4 M NaCl, 20 mM EDTA, 100 mM Tris.
2. Chloroform/isoamyl alcohol (24:1).
3. 75% and absolute ethanol.
4. Ultra-pure and sterile water (ddH 2 O).
5. Liquid nitrogen.
6. 1.5 mL Microcentrifuge tubes.
7. Steel balls.
8. Homogenizer (e.g., Retsch, Mixer Mill MM 400).
9. 65
C Incubator.
10. Centrifuge.
11. TaKaRa Taq DNA Polymerase.
12. PCR primer sets (10 μM) (Fig. 3b, see Table 1).
13. Thermocycler.
14. Restriction enzyme and reaction buffer (Fig. 3b).
15. Agarose, loading buffer, and TAE.
16. Agarose gel electrophoresis tank and power supply.
2.5 Generation of
Gene Targeting
Constructs
1. DD45 pro::Cas9 parental line (CS69955 and CS69956 from
Arabidopsis Resource Center (ABRC)).
2. Binary T-DNA vector pCAMBIA3301 (alternatively other
T-DNA vector harboring Basta resistance gene).
3. High-fidelity polymerase (e.g., KOD DNA polymerase, EMB
Millipore, #71085), reaction buffer, and dNTPs.
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