3. Spectrofluorometer.
4. Transmission Electron Microscope (TEM).
5. Nanoparticle analyzer, e.g., Nanosight NS500.
3 Methods
All procedures can be conducted on the bench top at room temperature, unless otherwise required.
3.1 Agarose
Gels (1%)
1. Suspend 0.2 g agarose in 18 mL ultrapure H 2 O and boil to
dissolve (see Note 5).
2. Cool to approximately 75
C and then add 2 mL of 10Â
electrophoresis buffer.
3. At this point, ethidium bromide (1 μg/mL) can be included in
the gel.
4. Place gel tray into a gel-casting tray. If no casting tray is available, then seal the gel tray with tape.
5. Add a multiwell comb about 1 cm away from the end of
the tray.
6. Once the gel has cooled to 60–62
C, pour into the gel tray and
allow gel to set for 1 h or less.
3.2 Electrophoretic
Mobility Shift
1. Use the 1% (for DNA) or 2% (for RNA) agarose gel as outlined
in Subheading 3.1.
2. While the gel is setting, prepare the nanocomplexes by using
varying amounts of the cationic nanoparticle and a constant
amount of the DNA/RNA (0.25 or 0.5 μg/μL) (see Note 6).
3. Incubate the nanocomplexes at room temperature for
20–30 min. However, for some functionalized or cationized
inorganic nanoparticle-based carriers such as gold, an incubation period of 1 h may be required [20].
4. Following the incubation period, add 1–2 μL of the gel loading
buffer to the nanocomplexes.
5. Load the nanocomplexes into the respective wells in the agarose gel. Include a naked nucleic acid control in one well (see
Note 7).
6. Place the loaded gel into an electrophoresis apparatus containing 1Â electrophoresis buffer (see Note 8).
7. Conduct the electrophoresis at 50 V for 1 h for DNA [21] and
for 30 min for RNA [22] molecules.
8. Remove the gel from the apparatus and view and obtain images
under UV transillumination at 300 nm (see Note 9).
9. A typical gel result for DNA is depicted in Fig. 1 (see Note 10).
46
Moganavelli Singh
4. Transmission Electron Microscope (TEM).
5. Nanoparticle analyzer, e.g., Nanosight NS500.
3 Methods
All procedures can be conducted on the bench top at room temperature, unless otherwise required.
3.1 Agarose
Gels (1%)
1. Suspend 0.2 g agarose in 18 mL ultrapure H 2 O and boil to
dissolve (see Note 5).
2. Cool to approximately 75
C and then add 2 mL of 10Â
electrophoresis buffer.
3. At this point, ethidium bromide (1 μg/mL) can be included in
the gel.
4. Place gel tray into a gel-casting tray. If no casting tray is available, then seal the gel tray with tape.
5. Add a multiwell comb about 1 cm away from the end of
the tray.
6. Once the gel has cooled to 60–62
C, pour into the gel tray and
allow gel to set for 1 h or less.
3.2 Electrophoretic
Mobility Shift
1. Use the 1% (for DNA) or 2% (for RNA) agarose gel as outlined
in Subheading 3.1.
2. While the gel is setting, prepare the nanocomplexes by using
varying amounts of the cationic nanoparticle and a constant
amount of the DNA/RNA (0.25 or 0.5 μg/μL) (see Note 6).
3. Incubate the nanocomplexes at room temperature for
20–30 min. However, for some functionalized or cationized
inorganic nanoparticle-based carriers such as gold, an incubation period of 1 h may be required [20].
4. Following the incubation period, add 1–2 μL of the gel loading
buffer to the nanocomplexes.
5. Load the nanocomplexes into the respective wells in the agarose gel. Include a naked nucleic acid control in one well (see
Note 7).
6. Place the loaded gel into an electrophoresis apparatus containing 1Â electrophoresis buffer (see Note 8).
7. Conduct the electrophoresis at 50 V for 1 h for DNA [21] and
for 30 min for RNA [22] molecules.
8. Remove the gel from the apparatus and view and obtain images
under UV transillumination at 300 nm (see Note 9).
9. A typical gel result for DNA is depicted in Fig. 1 (see Note 10).
46
Moganavelli Singh