Biology Reference
In-Depth Information
8. Glass microscope slides.
9. 1 % toluidine blue, 1 % sodium tetraborate in water.
10. Single hair attached to a wooden handle.
11. 200 mesh nickel EM grids (Veco), coated with 0.25 % Formvar
in chloroform.
1. Saturated uranyl acetate (8-10 % w/v in water). Protect from
light. Follow safety measures as described in previous
sections.
2. 0.2 % lead citrate in 0.4 % NaOH. Store at 4 °C and seal with
Parafilm to avoid formation of precipitates due to contact with
oxygen. Caution: avoid contact with eyes, skin and clothing.
Avoid ingestion and inhalation.
3. Whatman filter paper.
4. Access to a 100/120 kV transmission electron microscope
(JEOL-JEM 1100 or similar).
2.6.3 Section Staining
and Observation
3
Methods
1. Prepare a 100 μL sample of completely disrupted adenovirus
by heating it at 65 °C for 15 min (
see
Note 6
).
2. Mix PI into the sample and take an emission spectrum from
580 to 700 nm, exciting at 535 nm. Note the position and
intensity of the emission maximum.
3. Repeat
steps 1
-
2
with different virus and PI concentrations to
determine the conditions producing maximum fluorescence
emission. The PI fluorescence maximum when bound to
dsDNA should occur at approx. 617 nm emission wavelength.
4. Adjust the excitation and emission slit widths and
spectrofluorimeter recording conditions to avoid signal
saturation. This will be the maximum signal to be expected for
the experiment.
5. Take an emission spectrum in the same conditions for a sample
containing only buffer and PI at the concentration determined
in
step 3
(blank). This will be the basal data to use for later
subtraction. Note that different blank spectra may need to be
obtained if the experiment involves the use of different buffers
(e.g., if testing stability against high ionic strength, or against
acidification).
6. Take an emission spectrum of the adenovirus sample in phys-
iological conditions, using the virus and PI concentrations
determined in
step 3
. Record emission from 580 to 700 nm,
exciting at 535 nm. Subtract the blank emission spectrum
taken in
step 5
. The intensity value of the corrected spec-
3.1 Fluorescence
Spectroscopy
