5. A 1% gel can be used for DNA, but for RNA a 2% gel may be
required [22]. Double the quantities required for the agarose
in this case. If one desires a thicker gel, the volume can be
doubled, but do not forget to double all other amounts as well.
6. Preparation of nanocomplexes ultimately will depend on the
binding of the cationic nanocarrier to the nucleic acid, which in
turn depends on the relative density of positive and negative
charges on the respective components that will facilitate electrostatic interaction. Metallic and inorganic nanoparticles are
commonly functionalized with polymers such as chitosan,
poly-L-lysine, dendrimers, polyethyleneimine (PEI) which confer positive charges for nanocomplexation. When deciding on
ratios it is wise to start from at least a low (nanoparticle:nucleic
acid) 1:1 (w/w) ratio and increase the nanoparticle amounts by
at least 0.5 μg each time. Cationic polymers on their own will
naturally have lower binding ratios so expect to use less of the
polymer to bind the nucleic acid. Often mRNA and siRNA
nanocomplexes would have higher binding ratios than for
DNA-based nanocomplexes.
7. The naked DNA is used as it would migrate freely into the gel
and produce the expected bands for the supercoiled, circular,
and linear forms. Since the migration of this DNA will be used
as a control against which the migration of unbound DNA will
be compared, the naked DNA used must be of the same
concentration and of the same type, e.g., substituting a plasmid
DNA (e.g., pBR322 DNA) with calf thymus DNA as the naked
control, may skew the results.
8. For most mini-sub electrophoresis apparatus approximately
200–250 mL of 1Â electrophoresis buffer is needed. This
should be diluted from the 10Â electrophoresis buffer stock.
9. Dedicated gel documentation or imaging systems are available
where one can view and capture images. Do not view UV light
directly as it is harmful to the eyes. Wear UV protective glasses.
10. Nucleic acid that binds to the carrier will be retarded in the gel
and appear closer to the wells, while the DNA/RNA on its own
will migrate into the gel. When all the negative charges on the
nucleic acid are completely bound to the positive charges of the
nanocarrier, an electroneutral complex is produced, that
remains in the well (Fig. 1, lanes 4 and 5). This is evident by
the bright fluorescence seen in the wells. This is regarded as the
optimum binding ratio (lane 4). The ratio above and ratio
below this are referred to as the supra-optimum (lane 5) and
sub-optimum (lane 3) ratios, respectively. We usually use these
three ratios for all subsequent evaluations in vitro. This assay is
also known as the band shift or gel retardation assay.
52
Moganavelli Singh
required [22]. Double the quantities required for the agarose
in this case. If one desires a thicker gel, the volume can be
doubled, but do not forget to double all other amounts as well.
6. Preparation of nanocomplexes ultimately will depend on the
binding of the cationic nanocarrier to the nucleic acid, which in
turn depends on the relative density of positive and negative
charges on the respective components that will facilitate electrostatic interaction. Metallic and inorganic nanoparticles are
commonly functionalized with polymers such as chitosan,
poly-L-lysine, dendrimers, polyethyleneimine (PEI) which confer positive charges for nanocomplexation. When deciding on
ratios it is wise to start from at least a low (nanoparticle:nucleic
acid) 1:1 (w/w) ratio and increase the nanoparticle amounts by
at least 0.5 μg each time. Cationic polymers on their own will
naturally have lower binding ratios so expect to use less of the
polymer to bind the nucleic acid. Often mRNA and siRNA
nanocomplexes would have higher binding ratios than for
DNA-based nanocomplexes.
7. The naked DNA is used as it would migrate freely into the gel
and produce the expected bands for the supercoiled, circular,
and linear forms. Since the migration of this DNA will be used
as a control against which the migration of unbound DNA will
be compared, the naked DNA used must be of the same
concentration and of the same type, e.g., substituting a plasmid
DNA (e.g., pBR322 DNA) with calf thymus DNA as the naked
control, may skew the results.
8. For most mini-sub electrophoresis apparatus approximately
200–250 mL of 1Â electrophoresis buffer is needed. This
should be diluted from the 10Â electrophoresis buffer stock.
9. Dedicated gel documentation or imaging systems are available
where one can view and capture images. Do not view UV light
directly as it is harmful to the eyes. Wear UV protective glasses.
10. Nucleic acid that binds to the carrier will be retarded in the gel
and appear closer to the wells, while the DNA/RNA on its own
will migrate into the gel. When all the negative charges on the
nucleic acid are completely bound to the positive charges of the
nanocarrier, an electroneutral complex is produced, that
remains in the well (Fig. 1, lanes 4 and 5). This is evident by
the bright fluorescence seen in the wells. This is regarded as the
optimum binding ratio (lane 4). The ratio above and ratio
below this are referred to as the supra-optimum (lane 5) and
sub-optimum (lane 3) ratios, respectively. We usually use these
three ratios for all subsequent evaluations in vitro. This assay is
also known as the band shift or gel retardation assay.
52
Moganavelli Singh