13. Do not breath directly onto the uranyl acetate solution, as this
can cause turbidity in the solution. This solution will be used
for negative staining for electron microscopy.
14. To prevent premature enzyme activity.
15. There are two types of buffer for Phusion DNA polymerase.
For this experiment, use only the 5Â HF buffer.
16. Methylene blue is used to stain the DNA band on the preparative gel to reduce DNA damage.
17. Restriction enzymes are normally supplied with the appropriate
buffer.
18. Competent cell preparation requires aseptic technique to avoid
contamination.
19. A colony (or part of the master plate streak) can be used
directly as a template in PCR by using a toothpick to touch
the colony (or streak) and then swirling the toothpick in the
PCR solution, followed by starting the reaction.
20. Plasmid verification should be undertaken by sequencing the
entire insert using an in house or commercial sequencing
service.
21. Transfection into HEK293T/17 cells is effective using the
calcium phosphate method.
22. The protocol describes the conditions for one transfection. In
practice, we generated seven transfections per preparation and
pool the media.
23. To avoid cells detaching from the culture plate.
24. To remove cell debris and other small components from the
culture media. Filtration must be performed gently to avoid
membrane shearing.
25. Gently release each concentration of sucrose onto the wall of
the UC tube to make sure the layers do not mix.
26. The 10% sucrose must fill to the top of the tube with no air
bubbles inside the tube to prevent the tube from cracking.
Balancing the UC tube with a counter weight is essential.
27. Do not set the brake for this step to prevent band disruption.
28. For determination of Zika VLP by EM, the purified VLP must
be stored at 4
C. If stored at À20
C, there might be damage
to the VLP structure by ice crystal formation.
Acknowledgments
This work was supported by Mahidol University, the Thailand
Research Fund (BRG6080006) and the Newton Fund as administered by the National Science and Technology Development
Agency (FDA-CO-2561-6820-TH).
202
Atichat Kuadkitkan et al.
can cause turbidity in the solution. This solution will be used
for negative staining for electron microscopy.
14. To prevent premature enzyme activity.
15. There are two types of buffer for Phusion DNA polymerase.
For this experiment, use only the 5Â HF buffer.
16. Methylene blue is used to stain the DNA band on the preparative gel to reduce DNA damage.
17. Restriction enzymes are normally supplied with the appropriate
buffer.
18. Competent cell preparation requires aseptic technique to avoid
contamination.
19. A colony (or part of the master plate streak) can be used
directly as a template in PCR by using a toothpick to touch
the colony (or streak) and then swirling the toothpick in the
PCR solution, followed by starting the reaction.
20. Plasmid verification should be undertaken by sequencing the
entire insert using an in house or commercial sequencing
service.
21. Transfection into HEK293T/17 cells is effective using the
calcium phosphate method.
22. The protocol describes the conditions for one transfection. In
practice, we generated seven transfections per preparation and
pool the media.
23. To avoid cells detaching from the culture plate.
24. To remove cell debris and other small components from the
culture media. Filtration must be performed gently to avoid
membrane shearing.
25. Gently release each concentration of sucrose onto the wall of
the UC tube to make sure the layers do not mix.
26. The 10% sucrose must fill to the top of the tube with no air
bubbles inside the tube to prevent the tube from cracking.
Balancing the UC tube with a counter weight is essential.
27. Do not set the brake for this step to prevent band disruption.
28. For determination of Zika VLP by EM, the purified VLP must
be stored at 4
C. If stored at À20
C, there might be damage
to the VLP structure by ice crystal formation.
Acknowledgments
This work was supported by Mahidol University, the Thailand
Research Fund (BRG6080006) and the Newton Fund as administered by the National Science and Technology Development
Agency (FDA-CO-2561-6820-TH).
202
Atichat Kuadkitkan et al.