2. The forward and reverse primers are NheI-19CprME-Zika-Fw
(5
0 -GAGCTAGCCACCATGGGAAGAGGGACCGATACAA
GC-3
0 ) and 19CprME-Zika-EcoRI-Rw (5
0 -CGGAATTCTTA
TGCGGACACTGCGGTGGACAGAAAA-3
0 ), respectively.
The plasmid construction has been briefly described in a previous study [13].
3. Phusion High-Fidelity DNA polymerase DreamTaq DNA
polymerase.
4. 10 mM dNTPs.
5. T4 DNA ligase enzyme.
6. 50Â TAE buffer: 2 M Tris–HCl, 1 M acetic acid, 0.1 M EDTA.
Weigh 242 g of Tris base and 37.2 g of ethylenediaminetetraacetic acid (EDTA) disodium salt, dihydrate (EDTA ∙ Na 2 ∙ 2H 2 O,
MW 372.24 g/mol) into a glass beaker and dissolve in 500 mL
of deionized water using a magnetic stirrer. After all the chemicals have completely dissolved, add 57.1 mL of glacial acetic
acid (see Note 2) to the solution and adjust the volume to 1 L
with deionized water. The stock buffer can be kept at room
temperature.
7. 1Â TAE buffer: add 20 mL 50Â TAE buffer to a 1 L measuring
cylinder and add deionized water to 1 L.
8. 1% agarose gel in 1Â TAE buffer. Weigh 1 g of agarose (molecular biology grade) and add 100 mL of 1Â TAE buffer into a
glass bottle then heat the agar in a microwave oven (see Note 3)
until it is completely melted. Let the melted agarose cool down
to 42
C followed by pouring into a gel setting chamber and
leave at room temperature for 30 min before use.
9. 0.5 μg/mL ethidium bromide (EthBr) DNA staining solution.
Take 15 μL of 10 mg/mL stock ethidium bromide solution in
water and mix with 300 mL of deionized water in a plastic
chamber (see Note 4).
10. 100 mg/mL (w/v) ampicillin stock. Weigh 1 g of ampicillin
powder into a glass beaker and dissolve in 8 mL of deionized
water using a magnetic stirrer. After the ampicillin has
completely dissolved, add deionized water to a final volume
of 10 mL. Then filter the ampicillin stock solution through a
0.22 μm PES membrane filter and aliquot into 1.5 mL tubes
for storage at À20
C.
11. LB broth. Weigh 4 g of tryptone powder, 4 g of sodium
chloride and 2 g of yeast extract into a glass bottle and add
400 mL of deionized water and mix thoroughly before autoclaving. Add 200 μL of stock ampicillin to a final concentration
of 50 μg/mL before use (see Note 5) and keep the
LB-ampicillin broth at 4
C.
Zika Virus-Like Particles
185
(5
0 -GAGCTAGCCACCATGGGAAGAGGGACCGATACAA
GC-3
0 ) and 19CprME-Zika-EcoRI-Rw (5
0 -CGGAATTCTTA
TGCGGACACTGCGGTGGACAGAAAA-3
0 ), respectively.
The plasmid construction has been briefly described in a previous study [13].
3. Phusion High-Fidelity DNA polymerase DreamTaq DNA
polymerase.
4. 10 mM dNTPs.
5. T4 DNA ligase enzyme.
6. 50Â TAE buffer: 2 M Tris–HCl, 1 M acetic acid, 0.1 M EDTA.
Weigh 242 g of Tris base and 37.2 g of ethylenediaminetetraacetic acid (EDTA) disodium salt, dihydrate (EDTA ∙ Na 2 ∙ 2H 2 O,
MW 372.24 g/mol) into a glass beaker and dissolve in 500 mL
of deionized water using a magnetic stirrer. After all the chemicals have completely dissolved, add 57.1 mL of glacial acetic
acid (see Note 2) to the solution and adjust the volume to 1 L
with deionized water. The stock buffer can be kept at room
temperature.
7. 1Â TAE buffer: add 20 mL 50Â TAE buffer to a 1 L measuring
cylinder and add deionized water to 1 L.
8. 1% agarose gel in 1Â TAE buffer. Weigh 1 g of agarose (molecular biology grade) and add 100 mL of 1Â TAE buffer into a
glass bottle then heat the agar in a microwave oven (see Note 3)
until it is completely melted. Let the melted agarose cool down
to 42
C followed by pouring into a gel setting chamber and
leave at room temperature for 30 min before use.
9. 0.5 μg/mL ethidium bromide (EthBr) DNA staining solution.
Take 15 μL of 10 mg/mL stock ethidium bromide solution in
water and mix with 300 mL of deionized water in a plastic
chamber (see Note 4).
10. 100 mg/mL (w/v) ampicillin stock. Weigh 1 g of ampicillin
powder into a glass beaker and dissolve in 8 mL of deionized
water using a magnetic stirrer. After the ampicillin has
completely dissolved, add deionized water to a final volume
of 10 mL. Then filter the ampicillin stock solution through a
0.22 μm PES membrane filter and aliquot into 1.5 mL tubes
for storage at À20
C.
11. LB broth. Weigh 4 g of tryptone powder, 4 g of sodium
chloride and 2 g of yeast extract into a glass bottle and add
400 mL of deionized water and mix thoroughly before autoclaving. Add 200 μL of stock ampicillin to a final concentration
of 50 μg/mL before use (see Note 5) and keep the
LB-ampicillin broth at 4
C.
Zika Virus-Like Particles
185