A. Raza et al. / Bioorg. Med. Chem. Lett. 23 (2013) 620–623
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10 mM of compound in the outer chamber. Degassed water was used as a
control in the outer chamber. All windows and the partitions were polished
1
and
3 sides wrapped with black electrical tape, leaving only one side
1
transparent to UV light. This arrangement allows the incident UV radiation
go through the protective solution (outer chamber) and onto DNA solution
(inner chamber). The tandem cuvettes were then exposed to UV radiation (kmax
300 nm, 35 W, Rayonet UV reactor, Southern Ultraviolet Co., Branford, CT) at
4 °C. Aliquots were then taken of the DNA solution at each time point (0, 10, 20,
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for thymidine dimer damage. The DNA was incubated for 1 h at 37 °C in the
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presence of 20 U (1 lL) T4 Endonuclease V and then analyzed by gel
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UV protection assay of plasmid DNA using acyclothymidine dinucleosides in cream
vehicle: A small amount of the cream (0.1 cc) was applied to the outside of
‘black’ 3 mL quartz cuvettes and a quartz slide, cut to the size of the cuvette,
was placed over the cream to make an even layer. DNA solution was placed in
the chamber and protected by using cream alone or compound in cream (5% w/
w). The quartz cuvette is wrapped on 3 sides with black electrical tape and
irradiated as described earlier. Aliquots of protected DNA solution were then
taken at 0, 10, 20, 30, 45, 60, 90 and 120 min intervals and analyzed by
enzymatic digestion and gel electrophoresis as previously stated.
UV protection assay of cellular DNA using acyclothymidine dinucleosides in cream
vehicle: RPMI-8226 cells (ATCC; Manassas, VA; 2.5 Â 106 cells/mL) were placed
in standard 3 mL quartz ‘black’ cuvettes (n = 3) with a stir bar in the bottom of
each cuvette to keep the cells in uniform suspension during the experiment. A
small amount of the cream (0.1 cc) was applied to the outside of the cuvette
and a quartz slide, cut to the size of the cuvette, was placed over the cream to
make an even layer. The quartz cuvette is wrapped on 3 sides with black
electrical tape. The cells were protected by using cream alone or compound in
cream (5% w/w) and irradiated as described earlier under continuous stirring
using a stir plate. At each time point, a cuvette containing cells was removed
from the UV reactor. Post UV exposure, the cells are transferred to sterile
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1. Sambrook, J.; Russell, W. D. Molecular Cloning; Cold Spring Harbor Laboratory
Press: Cold Spring Harbor, 2001; Chapter 6.
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3
1.7 mL tubes and the cuvettes are washed with PBS (500 lL) and the contents
3
2. UV protection assay of plasmid DNA using acyclothymidine dinucleosides in
aqueous solution: Tandem quartz cuvettes (NSG Precision Cells Farmingdale,
NY; 1 cm  3 cm) were used in DNA photoprotection assays. These cuvettes
consist of two internal chambers (ꢀ1.5 mL each). The inner chamber (0.5 cm
path length) was filled with an aqueous solution of DNA (pEGFP-C1 plasmid
transferred to the corresponding sterile 1.7 mL tube. The tubes were
centrifuged (13,000 rpm, 2 min, 4 °C), supernatant decanted and the cell
pellets re-suspended in Proteinase K solution. The tubes were incubated for 3 h
at 55 °C and the DNA isolated using standard DNA isolation protocol. The
isolated DNA was verified by enzymatic digestion and gel electrophoresis as
previously stated.
31
DNA, Clontech/Takara, Mountain View, CA; 100 lg) and was protected by