6. LB-ampicillin and LB-kanamycin: 25 g/L of Difco™ LB broth
and 100 μg/mL ampicillin or 50 μg/mL kanamycin.
7. LB-ampicillin agar and LB-kanamycin agar: 35 g/L of LB agar,
Lennox and 100 μg/mL ampicillin, or 50 μg/mL kanamycin.
8. 100 μg/mL Ampicillin.
9. 50 μg/mL Kanamycin.
10. 100% Glycerol.
11. 250-mL Erlenmeyer shake flasks.
12. Sterile pipette tips (2, 200, and 1000 μL).
13. 1.5-mL Eppendorf tubes.
14. Sterile cell spreader.
15. Tabletop centrifuge.
16. Incubator with shaker.
17. Plasmid mini- and midiprep kits.
18. 0.7% agarose gel: 0.7 g agarose powder dissolved in 100 mL
1Â TBE buffer.
19. Novel juice (DNA staining reagent) (GeneDireX).
20. Standard ladder Lambda DNA/HindIII Marker (Thermo
Fisher Scientific).
21. Standard ladder 1 Kb plus DNA ladder (Thermo Fisher
Scientific).
22. Gel chamber and power source.
23. Gel documentation machine.
24. NanoDrop 8000 (Thermo Fisher Scientific).
25. Water bath.
26. Ice box.
2.3 Transfection
of CRISPR/Cas9
and GFP Constructs
into K562 and Raji
Cell Lines
1. K562 and Raji cell culture medium: RPMI-1640, 10% FBS,
2 mM GlutaMAX™, 100 U/mL Penicillin-Streptomycin.
2. Purified sgRNA plasmid (gRNA_AAVS1-T2), Cas9 plasmid
(hCas9), and GFP donor plasmid (AAV-CAG-EGFP).
3. P3 primary cell 4D-nucleofector X kit (Lonza).
4. 100 μL single Nucleocuvette™ (supplied with the kit).
5. Zombie Aqua™ Fixable Viability Kit (Biolegend).
6. 24-well plate.
7. 1.5-mL Eppendorf tubes.
8. Hemocytometer.
9. Sterile seropipettes, pipette tips (2, 200, and 1000 μL).
10. Inverted microscope.
11. Tabletop centrifuge.
216
Nontaphat Thongsin and Methichit Wattanapanitch
and 100 μg/mL ampicillin or 50 μg/mL kanamycin.
7. LB-ampicillin agar and LB-kanamycin agar: 35 g/L of LB agar,
Lennox and 100 μg/mL ampicillin, or 50 μg/mL kanamycin.
8. 100 μg/mL Ampicillin.
9. 50 μg/mL Kanamycin.
10. 100% Glycerol.
11. 250-mL Erlenmeyer shake flasks.
12. Sterile pipette tips (2, 200, and 1000 μL).
13. 1.5-mL Eppendorf tubes.
14. Sterile cell spreader.
15. Tabletop centrifuge.
16. Incubator with shaker.
17. Plasmid mini- and midiprep kits.
18. 0.7% agarose gel: 0.7 g agarose powder dissolved in 100 mL
1Â TBE buffer.
19. Novel juice (DNA staining reagent) (GeneDireX).
20. Standard ladder Lambda DNA/HindIII Marker (Thermo
Fisher Scientific).
21. Standard ladder 1 Kb plus DNA ladder (Thermo Fisher
Scientific).
22. Gel chamber and power source.
23. Gel documentation machine.
24. NanoDrop 8000 (Thermo Fisher Scientific).
25. Water bath.
26. Ice box.
2.3 Transfection
of CRISPR/Cas9
and GFP Constructs
into K562 and Raji
Cell Lines
1. K562 and Raji cell culture medium: RPMI-1640, 10% FBS,
2 mM GlutaMAX™, 100 U/mL Penicillin-Streptomycin.
2. Purified sgRNA plasmid (gRNA_AAVS1-T2), Cas9 plasmid
(hCas9), and GFP donor plasmid (AAV-CAG-EGFP).
3. P3 primary cell 4D-nucleofector X kit (Lonza).
4. 100 μL single Nucleocuvette™ (supplied with the kit).
5. Zombie Aqua™ Fixable Viability Kit (Biolegend).
6. 24-well plate.
7. 1.5-mL Eppendorf tubes.
8. Hemocytometer.
9. Sterile seropipettes, pipette tips (2, 200, and 1000 μL).
10. Inverted microscope.
11. Tabletop centrifuge.
216
Nontaphat Thongsin and Methichit Wattanapanitch