School of Engineering and Science - Jacobs University

Material and Methods
4.2.6.2 Alkaline transfer of DNA
The DNA for Southern blot analysis was separated by gelelectrophoresis
(4.2.3) on a 0.8% agarose gel. After documentation of the gel, the DNA was
transferred to a positively charged nylon membrane (Hybond N + ) by capillary
transfer using 5x SSC/10 mM NaOH buffer. The transfer was performed over
night. After transfer the DNA was fixed to the membrane using a UVcrosslinker.
The dried membrane could be stored at room temperature until
hybridisation.
Composition of 20x SSC is 3 M NaCl and 0.3 M tri-sodium citrate.
4.2.6.3 Hybridisation and detection
Prior to hybridisation with the probe, the membrane was incubated with
hybridisation buffer (0.1% n-laurylsarcosin, 0.02% SDS and 1% blocking
reagent in 5x SSC) for 1 hour. In the meantime, the DNA-probe was
denaturated for 10 min. at 95°C and then shock-cooled in an ethanol/ice bath.
Then the probe was resuspended in 10 ml of pre-warmed hybridisation buffer.
Then the probe/buffer mixture was added to the membrane and hybridised for
12-16 hours at the respective temperature. After hybridisation the membrane
was washed two times under non-stringent conditions (5 min. room
temperature in 2x SSC, 0.1% SDS) and two times under stringent conditions
(15 min at hybridisation temperature in 0.1x SSC, 0.1% SDS). Thereafter the
membrane was equilibrated at room temperature for 1 min. in buffer 1 (100 mM
Tris/HCl at pH 7.5 with 150 mM NaCl). Then the membrane was incubated at
room temperature for 30 min. in the blocking buffer 2 (0.5% blocking reagent in
buffer 1). After a washing the membrane in buffer 1, the antibody solution (2 µl
of anti-digoxygenin-ap, fab-fragment by Roche, Mannheim, in 20 ml of buffer 1)
was added to the membrane and incubated at room temperature for 30 min..
Any excess of antibody was removed by two washing steps in buffer 1 (15 min.,
room temperature). Finally, the membrane was equilibrated for 1 min. in the
detection buffer (0.1 M Tris/HCL, 100 mM NaCl, and 50 mM MgCl 2 at an pH of
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