Detection and quantification of Damaged DNA. 12-day-old sterile cultured seedlings were
treated for 30 min with UV-B and then incubated in dark conditions. After three days the
seedlings were transferred to long-day conditions. After four more days the bleached phenotype
could be observed. For detection of UV-B photo damage, 12-day-old plants were subjected to
UV-B for 30 min and directly incubated under dark conditions. Samples were taken immediately
following the irradiation and after 72h of incubation. The genomic DNA was extracted using
standard
procedures.
The
DNA
concentration
was
photometrically
measured
and
densitometrically equalized using the image analysis software ImageJ (Abramoff et al., 2004).
Equal amounts of DNA were heat denatured and dot-blotted onto a nylon membrane (Nytran N,
Whatman). For each sample three aliquots were blotted to calculate the mean value. Crosslinking was done by baking the membranes at 80 °C for 2h. The membranes were blocked with
6% fat-free skim milk in T-PBS (50 mM Tris/HCl pH 8.0; 138 mM NaCl; 2.7 mM KCl; 0.1 %
Tween-20) for one hour, followed by three 10 min washing steps with T-PBS. Membranes were
incubated with the primary antibody for 2h. For detection of UV photo lesions a polyclonal
antibody was used derived from UV-irradiated polythymidylic acid treated rabbits (PA1-2000,
Affinity BioReagents). After three washing steps in T-PBS, the membranes were incubated with
the secondary horseradish peroxidase conjugated antibody (Santa Cruz Biotechnology, Inc, Santa
Cruz, CA, USA) for 2h. The membranes were washed five times for 10 min before the
application of the chemiluminescent reagents (ECL Plus Western Blotting Detection System,
Amersham) and the subsequent detection of the fluorescence signal in a Typhoon 9400
phosphorimager (GE healthcare). The data analysis was performed using ImageJ. Experiments
were replicated three to five times.