This is the world where the talent is supposed to be allowed to unwind and really relax after dealing with reporters and assorted taggers-along all evening.
On the night I tried it, I only wished that the spaghetti had had a little more snap; sometimes the pasta station here seems to lose track of time, and a bowl of noodles that should be tensely coiled is allowed to unwind just a bit.
Next, slides were transferred to a horizontal electrophoresis tray (Owl separation system) filled with an alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) and DNA was allowed to unwind during 30 min at 4°C.
In this assay, cells are broken apart to remove proteins, and the DNA is allowed to unwind.
The slides were then placed in an electrophoresis tank, and the DNA was allowed to unwind for 15 min in the alkaline solution.
DNA was allowed to unwind in alkaline conditions (300 mM NaOH, 1 mM Na2EDTA·2H2O, pH > 13) for 40 min before subjecting to electrophoresis for 30 min at 0.7 V/cm.
Subsequently, the DNA was allowed to unwind in electrophoresis solution (0.3 M NaOH and 1 mM Na2EDTA, pH 13) for 40 min at 4°C before electrophoresis at 0.9 V/cm, 300 mA for 30 min at 4°C.
After the lysis, the slides were placed in an electrophoresis unit, DNA was allowed to unwind for 20 min in the solution consisting of 300 mM NaOH and 1 mM EDTA, pH > 13.
DSBs are detected by an N/N assay when pH of lysing and electrophoretic solutions is kept under pH 10, whilst for the detection of SSBs DNA after the lysis is allowed to unwind DNA double-helix in alkali [ 17] to separate individual strands and expose their fragmentation (A/N protocol [ 9]).
After overnight lysis (+4°C) in alkaline buffer (pH 10) of cellular and nuclear membranes, the slides will be placed in a horizontal electrophoresis box, allowed to unwind for 20 min in an electrophoretic alkaline buffer (pH > 13) and then subjected to electrophoresis (+4°C) for 20 min by applying an electric field of 1 V/cm and adjusting the current to 300 mA.
The tank was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM Na2 EDTA), and the DNA was allowed to unwind for 40 min before electrophoresis, which was performed at 25 V for 30 min. After electrophoresis the slides were washed 3 times, for 5 min each time, in neutralizing buffer (0.4 M Tris, pH 7.5).
