Determining the Cellular Targets of Reactive Oxygen Species in Borrelia burgdorferi - Bacterial Pathogenesis: Methods and Protocols - page 205

4. Isolate total genomic DNA using DNeasy tissue kit (Qiagen) and quantify.
5. Use primers that will generate fragments 5-10 kb in length and perform PCR
using an Expand Long Template PCR system (Roche). Briefly, the 25-μL PCR
reaction mixture should contain 0.1 ng of genomic DNA as a template, 300 n M
of each primer, 350 μ M of dNTPs, 1× PCR buffer containing 0.175 m M MgCl 2 ,
and 0.75 μL of DNA polymerase.
6. Separate products on a 1% agarose gel, stain with ethidium bromide, and visualize
using UV.
3.3. Quantitation of DNA Damage
1. Treat cells and isolate DNA as described above.
2. Dilute DNA to 0.1 mg/mL in PBS.
3. Combine 5 μL of diluted DNA with 5 μL of 10 m M ARP reagent (Oxford
Biomedical Research) and incubate for1hat37°C.
4. To precipitate the DNA, combine 88 μL of Tris-EDTA, 2 μL of 10 mg/mL
glycogen, and the DNA-ARP mixture. Add 300 μL of cold ethanol and mix
well. Cap and store at -20 °C for 10 min. Centrifuge the samples at 14,000 × g
for 10 min and discard the supernatant. Wash the pellet thrice with 0.5 mL
of 70% ethanol. Allow the pellet to dry for 3-5 min. Dissolve DNA pellet in
Tris-EDTA. Determine the concentration using a spectrophotometer and add
Tris-EDTA to make a final concentration of 0.5 μg/mL.
5. In triplicate, pipet 60 μL of sample into one well of a 96-well microplate. The
microplateshouldbeprewashedwithTPBS4×, followedby twowasheswithdH 2 O.
6. Cover the plate and incubate at 37 °C overnight.
7. After the incubation, wash the plate 4× with TPBS.
8. Dilute the HRP-streptavidin conjugate (Invitrogen) to 0.5 μg/mL in assay buffer
and pipet 100 μL to each well. Shake plate at 100 rev/min on a platform shaker
for1hatroom temperature.
9. Wash wells five times with TPBS.
10. Add 100 μL of TMB single solution (Invitrogen) to each well and incubate for
1hat37°C.
11. To quench, add 100 μL of 1 M H 2 SO 4 and read at 450 nm. Quantitate using ARP
standards (Oxford Biomedical Research). Graph the standard curve by plotting
ARP versus OD.
3.4. Identification of Lipid Damage
1. Grow and treat cells as described above.
2. Centrifuge2×10 7 cells at 1000 × g for 5 min at room temperature.
3. Wash cells 2× with HEPES/NaCl buffer.
4. Resuspend cells in 1 mL of diluent C.
5. Add 4 μL of red flourescent dye to 1 mL diluent C.
6. Add 1 mL of cell suspension to 1 mL of diluted dye. Incubate for 5 min at room
temperature.