4. To fresh PCR product (0.5–4 μl) add 1 μl of salt solution,
sterile water to a final volume of 5 μl, and 1 μl of the TOPO
®
vector.
5. Mix gently and incubate for 5 min at room temperature.
6. Place on ice and proceed to transform One Shot
® TOP10
chemically competent E. coli.
7. Add 3 μl of the TOPO
® cloning reaction into a vial of One
Shot
® TOP10 chemically competent E. coli and mix gently.
8. Incubate on ice for 5–30 min.
9. Heat-shock the cells for 30 s at 42
C without shaking.
10. Immediately transfer the tube to ice.
11. Add 250 μl of room-temperature S.O.C. medium.
12. Incubate at 37
C for 1 h with shaking, then spread 100–200 μl
of bacterial culture on a prewarmed selection plate and incubate overnight at 37
C.
13. Select ten colonies to test the gene product by PCR.
14. Grow the TOP10 E. coli overnight in LB medium.
15. Purify the plasmid using a plasmid purification kit.
16. Transform BL21 Star™(DE3) with synthetic EFn-3ÂM2eHA2-containing plasmid.
17. Check the gene product by PCR.
18. Select the best colony and grow in 20 ml of LB liquid medium
overnight.
19. Transfer the 20 ml of bacteria culture to 4 L of LB, incubate for
2–4 h at 37
C, and read the absorbance at 620 nm. The
absorbance should be in the range 0.4–0.6 before adding
IPTG (0.05 mM).
20. Grow the culture for 6 h at 30
C.
21. Centrifuge the bacteria at 10,000 Â g for 10 min at 4
C. Place
the bacterial pellet on ice.
22. Dilute the bacterial pellet by adding 5–10 ml of binding buffer
(0.5 M NaCl, 40 mM imidazole, and 8 M urea) for each gram
of cell paste.
23. Disrupt cells by sonication (in several short bursts) on ice for
approximately 10 min.
24. Centrifuge the bacteria at 15,000 Â g for 30 min at 4
C.
25. Collect the supernatant and filtrate using a Minisart NY25
0.45-μm filter.
26. Equilibrate a Ni-NTA Superflow column using binding buffer
for 30 min.
27. Load the lysed bacteria onto the column.
Vaccine Delivery with a Detoxified Bacterial Toxin
429
sterile water to a final volume of 5 μl, and 1 μl of the TOPO
®
vector.
5. Mix gently and incubate for 5 min at room temperature.
6. Place on ice and proceed to transform One Shot
® TOP10
chemically competent E. coli.
7. Add 3 μl of the TOPO
® cloning reaction into a vial of One
Shot
® TOP10 chemically competent E. coli and mix gently.
8. Incubate on ice for 5–30 min.
9. Heat-shock the cells for 30 s at 42
C without shaking.
10. Immediately transfer the tube to ice.
11. Add 250 μl of room-temperature S.O.C. medium.
12. Incubate at 37
C for 1 h with shaking, then spread 100–200 μl
of bacterial culture on a prewarmed selection plate and incubate overnight at 37
C.
13. Select ten colonies to test the gene product by PCR.
14. Grow the TOP10 E. coli overnight in LB medium.
15. Purify the plasmid using a plasmid purification kit.
16. Transform BL21 Star™(DE3) with synthetic EFn-3ÂM2eHA2-containing plasmid.
17. Check the gene product by PCR.
18. Select the best colony and grow in 20 ml of LB liquid medium
overnight.
19. Transfer the 20 ml of bacteria culture to 4 L of LB, incubate for
2–4 h at 37
C, and read the absorbance at 620 nm. The
absorbance should be in the range 0.4–0.6 before adding
IPTG (0.05 mM).
20. Grow the culture for 6 h at 30
C.
21. Centrifuge the bacteria at 10,000 Â g for 10 min at 4
C. Place
the bacterial pellet on ice.
22. Dilute the bacterial pellet by adding 5–10 ml of binding buffer
(0.5 M NaCl, 40 mM imidazole, and 8 M urea) for each gram
of cell paste.
23. Disrupt cells by sonication (in several short bursts) on ice for
approximately 10 min.
24. Centrifuge the bacteria at 15,000 Â g for 30 min at 4
C.
25. Collect the supernatant and filtrate using a Minisart NY25
0.45-μm filter.
26. Equilibrate a Ni-NTA Superflow column using binding buffer
for 30 min.
27. Load the lysed bacteria onto the column.
Vaccine Delivery with a Detoxified Bacterial Toxin
429