A 3 kb ssDNA molecular construct was obtained by pH denaturation (strand separation) of a 3 kb dsDNA (see Fig. I.1). The dsDNA was obtained from PCR amplification of a 3 kbp fragment of -DNA. One of the primers used in the process was already labeled with Biotin. The resulting product was cleaved with the endonuclease XbaI producing a cohesive end. Another 24 base oligonucleotide (previously labeled with several digoxigenins at its end by using terminal transferase) was hybridized with a second 20-base long oligonucleotide giving a DNA construction with one cohesive end complementary to XbaI. Both products were annealed and ligated resulting in one 3 kbp dsDNA molecule.
To produce ssDNA, the molecular construct was incubated with Strepavidin coated beads for 30 min at room temperature in a volume of 15 l of 10 mM TE buffer. Afterwards, 35 l of 0.1 M [NaOH] were added in order to cause the separation (i.e., denaturation) of the strands. After 30 min, the sample was centrifuged. The white precipitate of beads and ssDNA was re-suspended in TE buffer. The second attachment with the antidigoxigenin beads was achieved in the fluidics chamber with the help of the micropipette (see Sec. 3.2.2).
JM Huguet 2014-02-12