solution overflows. Heat-resistant gloves should be worn at all
times when handling flasks containing hot agarose gel solution.
2. Hot agarose gel solution can be maintained in a 60
C water
bath. Alternatively, 40 mL of boiled agarose gel solution can be
left on the benchtop to cool down for 10–20 min at room
temperature.
3. Rationale of N/P ratio: The N/P ratio is a measurement of the
ionic balance of noncovalent CPP/DNA complexes in vitro. It
refers to the number of nitrogen residues of CPP per phosphate
of DNA. Approximately, 1 μg of SR9 peptide [11] is equivalent
to 7 nmol of cationic nitrogen. One μg of a 2000-bp plasmid
DNA is equivalent to 3 nmol of anionic phosphate. The N/P
ratio of an SR/DNA complex is 7 Ä 3 ¼ 2.3, for example, if
you mix 1 μg SR9 with 1 μg DNA together. There are two ways
to obtain a desired N/P ratio. Based on the N/P fraction, you
can set the denominator P constant and vary the numerator N,
or vice versa. (1) Nitrogen is defined by the number of the
primary cation group (–NH 3
+ ) of peptides. One μg of SR9
peptide is equivalent to 7 nmol, based on the calculation:
(1 Â 10
À6 ) g Ä 1423.7 g/mol  10 positive “net”
charges ≒ 7 Â 10
À9 mol ¼ 7 nmol. On the other hand, one
μg of L5a peptide is equivalent to 3.6 nmol, based on the
calculation: (1 Â 10
À6
) g Ä 830.95 g/mol  3 positive “net”
charges ≒ 3.6 Â 10
À9 mol ¼ 3.6 nmol. (2) Phosphate is
defined by the number of the primary anion group (–PO 4
À )
of nucleic acids. One μg of a 2000 bp plasmid DNA is equivalent to 3 nmol, based on the calculation: (1 Â 10
À6 ) g Ä (bp in
full-length  660) g/mol  (bp in full-length À1 + D)  2
strands ≒ 3 Â 10
À9 mol ¼ 3 nmol, where D represents the
digital number of primary phosphate group at the 5
0 end.
4. Two μg (7.4 μL) of the pEGFP-N1 plasmid with 4733 bp in
length is equivalent to 6 nmol. Ten μg (1 μL) of the L5a
peptide is equivalent to 36 nmol.
5. Other software can be used to quantify the green fluorescent
intensity of plasmid DNA stained by SYBR Safe, such as the
UN-SCAN-IT from Silk Scientific Inc. [8] and the public
domain ImageJ software (https://imagej.nih.gov/ij/) [10].
6. Rationale of gel retardation assay: Gel retardation assay also
known as electrophoretic mobility shift assay (EMSA) is an
affinity electrophoresis technique commonly used to study protein–nucleic acid interactions. This method can determine
whether a protein binds to a specific DNA sequence or not. In
the absence of protein, a DNA labeled with radioisotope or fluorescein reveals a single band on a polyacrylamide or agarose gel
during electrophoretic separation for a short period of time. This
single DNA band corresponds to unbound DNA fragment and
L5a CPP for Intracellular Delivery of DNA
119
times when handling flasks containing hot agarose gel solution.
2. Hot agarose gel solution can be maintained in a 60
C water
bath. Alternatively, 40 mL of boiled agarose gel solution can be
left on the benchtop to cool down for 10–20 min at room
temperature.
3. Rationale of N/P ratio: The N/P ratio is a measurement of the
ionic balance of noncovalent CPP/DNA complexes in vitro. It
refers to the number of nitrogen residues of CPP per phosphate
of DNA. Approximately, 1 μg of SR9 peptide [11] is equivalent
to 7 nmol of cationic nitrogen. One μg of a 2000-bp plasmid
DNA is equivalent to 3 nmol of anionic phosphate. The N/P
ratio of an SR/DNA complex is 7 Ä 3 ¼ 2.3, for example, if
you mix 1 μg SR9 with 1 μg DNA together. There are two ways
to obtain a desired N/P ratio. Based on the N/P fraction, you
can set the denominator P constant and vary the numerator N,
or vice versa. (1) Nitrogen is defined by the number of the
primary cation group (–NH 3
+ ) of peptides. One μg of SR9
peptide is equivalent to 7 nmol, based on the calculation:
(1 Â 10
À6 ) g Ä 1423.7 g/mol  10 positive “net”
charges ≒ 7 Â 10
À9 mol ¼ 7 nmol. On the other hand, one
μg of L5a peptide is equivalent to 3.6 nmol, based on the
calculation: (1 Â 10
À6
) g Ä 830.95 g/mol  3 positive “net”
charges ≒ 3.6 Â 10
À9 mol ¼ 3.6 nmol. (2) Phosphate is
defined by the number of the primary anion group (–PO 4
À )
of nucleic acids. One μg of a 2000 bp plasmid DNA is equivalent to 3 nmol, based on the calculation: (1 Â 10
À6 ) g Ä (bp in
full-length  660) g/mol  (bp in full-length À1 + D)  2
strands ≒ 3 Â 10
À9 mol ¼ 3 nmol, where D represents the
digital number of primary phosphate group at the 5
0 end.
4. Two μg (7.4 μL) of the pEGFP-N1 plasmid with 4733 bp in
length is equivalent to 6 nmol. Ten μg (1 μL) of the L5a
peptide is equivalent to 36 nmol.
5. Other software can be used to quantify the green fluorescent
intensity of plasmid DNA stained by SYBR Safe, such as the
UN-SCAN-IT from Silk Scientific Inc. [8] and the public
domain ImageJ software (https://imagej.nih.gov/ij/) [10].
6. Rationale of gel retardation assay: Gel retardation assay also
known as electrophoretic mobility shift assay (EMSA) is an
affinity electrophoresis technique commonly used to study protein–nucleic acid interactions. This method can determine
whether a protein binds to a specific DNA sequence or not. In
the absence of protein, a DNA labeled with radioisotope or fluorescein reveals a single band on a polyacrylamide or agarose gel
during electrophoretic separation for a short period of time. This
single DNA band corresponds to unbound DNA fragment and
L5a CPP for Intracellular Delivery of DNA
119