Biology Reference
In-Depth Information
4. Take up virus solution into syringe, ensure that solution is
bubble free, and inject virus into the cell until the glass section
is covered (
see
Note 7
).
5. Place block on microscope, ensure that the laser is on, and
open shutter.
6. Look for virus movement which will be noted as small dancing
particles in the green beam clearly (
see
Note 8
). The “sweet
spot” for visualizing virus is just to the right of the larger space
(
see
Note 9
).
7. Close shutter, click capture on the NTA software. Adjust the
gain/exposure and refocus.
8. Record for 60 s, polydispersity medium and concentration
medium (
see
Note 10
).
9. After movie has recorded, the gain can be adjusted again if
needs be. Autotracking option will focus on the center of each
particle. Once happy, click “Analyse” (
see
Note 11
).
10. Record three different parts of the beam and average results
(vp/mL).
11. Set report options and save/print using CutePDF (
see
Note 12
).
4
Notes
1. We normally use gel extraction kits from Promega or
Invitrogen, and fi nd both work well, with reasonable
recoveries.
2. As a general rule, our laboratory uses 293 cells for viral propa-
gation, however alternative E1 complementing cell lines can
be used, for example 911 cells or PERC6 cells.
3. Extensive notes on these protocols, giving full details are
found in our previous chapter—please
see
ref. [
25
].
4. Hypothetically, it is possible to fuse all three fragments directly
in one PCR reaction using all three PCR products in approx.
equimolar amounts, with the fl anking primers. However, we
have found it more successful to perform two separate PCR
reactions.
5. Our laboratory uses commercially available ladders from
Promega, which work well, however we assume that any main-
stream supplier should work equally well.
6. We normally make up a 2/3 mL working solution in case (as
occasionally happens) the run fails.
7. It aids visualization at this point to have the laser switched on.
8. The speed of movement indicates particle size, not how big
they look on screen.
