12. LB agar. Weigh 4 g of tryptone powder, 4 g of sodium chloride, 2 g of yeast extract, and 8 g of bacteriological agar into a
glass bottle, add 400 mL of deionized water, then mix thoroughly before autoclaving. After sterilization, let the melted
agar solution cool down to 42
C and then add 400 μL of stock
ampicillin to a final concentration of 100 μg/mL (see Note 6).
Then pour 10 mL of the melted agar into a Sterilin single-use
plastic plate; let the agar set and keep at 4
C.
13. 0.1 M CaCl 2 . Weigh 5.88 g of CaCl 2 powder into a glass bottle
and add 400 mL of deionized water; then mix thoroughly
before autoclaving and keep at 4
C.
14. 1 M CaCl 2 . Weigh 58.81 g of CaCl 2 powder into a glass bottle
and add 400 mL of deionized water; then mix thoroughly
before autoclaving and keep at 4
C.
15. 15% glycerol with 0.1 M CaCl 2 . Measure 60 mL of 100%
glycerol into a 500 mL measuring cylinder, add 40 mL of
1 M CaCl 2 and 300 mL of deionized water, then mix thoroughly before transferring into a glass bottle glass followed by
autoclaving and storage at 4
C.
16. 0.22 μm polyethersulfone (PES) membrane filter.
17. Sterilin Single-use plastic plates.
18. Gel electrophoresis chamber.
19. Microwave oven.
20. FavorPrep™ GEL/PCR Purification Mini Kit.
21. FavorPrep™ Plasmid Extraction Mini Kit.
22. NanoDrop™ 2000 spectrophotometer.
23. 1.7 mL microcentrifuge tubes.
24. 0.2 mL nuclease-free microcentrifuge tubes.
25. Microcentrifuge capable of centrifuging 0.2 mL and 1.7 mL
microcentrifuge tubes.
26. Methylene blue.
27. NheI FastDigest restriction enzyme with provided buffer.
28. EcoRI FastDigest restriction enzyme with provided buffer.
29. Escherichia coli (E. coli) strain DH5α.
30. Autoclaved (sterile) toothpicks.
31. 250 mL bacterial culture flask.
32. Corning 50 mL centrifuge tubes.
33. Shaking incubator.
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Atichat Kuadkitkan et al.
glass bottle, add 400 mL of deionized water, then mix thoroughly before autoclaving. After sterilization, let the melted
agar solution cool down to 42
C and then add 400 μL of stock
ampicillin to a final concentration of 100 μg/mL (see Note 6).
Then pour 10 mL of the melted agar into a Sterilin single-use
plastic plate; let the agar set and keep at 4
C.
13. 0.1 M CaCl 2 . Weigh 5.88 g of CaCl 2 powder into a glass bottle
and add 400 mL of deionized water; then mix thoroughly
before autoclaving and keep at 4
C.
14. 1 M CaCl 2 . Weigh 58.81 g of CaCl 2 powder into a glass bottle
and add 400 mL of deionized water; then mix thoroughly
before autoclaving and keep at 4
C.
15. 15% glycerol with 0.1 M CaCl 2 . Measure 60 mL of 100%
glycerol into a 500 mL measuring cylinder, add 40 mL of
1 M CaCl 2 and 300 mL of deionized water, then mix thoroughly before transferring into a glass bottle glass followed by
autoclaving and storage at 4
C.
16. 0.22 μm polyethersulfone (PES) membrane filter.
17. Sterilin Single-use plastic plates.
18. Gel electrophoresis chamber.
19. Microwave oven.
20. FavorPrep™ GEL/PCR Purification Mini Kit.
21. FavorPrep™ Plasmid Extraction Mini Kit.
22. NanoDrop™ 2000 spectrophotometer.
23. 1.7 mL microcentrifuge tubes.
24. 0.2 mL nuclease-free microcentrifuge tubes.
25. Microcentrifuge capable of centrifuging 0.2 mL and 1.7 mL
microcentrifuge tubes.
26. Methylene blue.
27. NheI FastDigest restriction enzyme with provided buffer.
28. EcoRI FastDigest restriction enzyme with provided buffer.
29. Escherichia coli (E. coli) strain DH5α.
30. Autoclaved (sterile) toothpicks.
31. 250 mL bacterial culture flask.
32. Corning 50 mL centrifuge tubes.
33. Shaking incubator.
186
Atichat Kuadkitkan et al.