Biomedical Engineering Reference
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transfected. Supercoiled DNA can enter cells more effi ciently than
relaxed plasmids during transfection (calcium phosphate) and fur-
thermore, surplus DNA can be toxic to the cells, highlighting the
requirement for quantifi cation of supercoiled DNA in addition to
that obtained from spectrophotometry. This is carried out by a gel
based quantifi cation method as follows:
1. Dilute plasmid DNA samples. Dilution is calculated from the
spectrophotometric reading (<1 mg/ml, 1:10 dilution;
between 1 and 4 mg/ml, 1:100 dilution; and >4 mg/ml
1:200 dilution).
2. Analyze samples by gel electrophoresis by running increasing
concentrations of each sample alongside equally increasing
concentrations of a 1 kb DNA ladder (500
ʼ
g/ml).
3. Prepare sample and ladder concentrations as presented in Fig.
1a
.
a
Sample
Plasmid DNA (µl)/
1kb ladder (µl)
Loadind dye
(µl)
dH
2
0
(µl)
Total
(µl)
0.5
1.0
1.5
2.0
0.5
1.0
1.5
2.0
3.0
3.0
3.0
3.0
16.5
16.0
15.5
15.0
20
20
20
20
b
Kilobases
Mass
(ng)
10.0 -
8.0 -
6.0 -
5.0 -
4.0 -
3.0 -
4
4
5
42
33
125
2.0 -
48
1.5 -
36
1.0 -
42
0.5 -
42
Fig. 1
Gel quantifi cation of supercoiled DNA. (
a
) Schematic presentation of DNA sample dilutions. Load 20
l of
each plasmid and ladder sample dilutions on 1 % agarose gel and allow running at 80 V. (
b
) Example of gel image
obtained from quantifi cation of pMD-RVGCVS24-B2c plasmid.
Left
: NEB 1 kb ladder;
Right
: Increasing concentra-
tions of ladder and plasmid (0.5-2.0
ʼ
l) samples were used to generate an average. Supercoiled DNA (
green box
)
is aligned to the closest ladder band (here 3 kb) and DNA concentration is quantifi ed by densitometry using equiva-
lent mass (ng) of the selected ladder band (here 125 ng). Non-supercoiled/relaxed DNA (
red box
) is omitted
ʼ
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