I. Synthesis of ssDNA

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 $ \sim$3 kbp fragment of $ \lambda $-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 $ 3'$ 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 $ \mu $l of 10 mM TE buffer. Afterwards, 35 $ \mu $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).

Figure I.1: Synthesis of the 3 kb dsDNA. The resulting dsDNA is obtained after pH denaturation. The ssDNA molecule can be stretched between two coated beads, since the Biotin and Digoxigenins labels are located on the same strand.
\includegraphics[width=\textwidth]{figs/appendix5/ssDNAsyn.eps}

JM Huguet 2014-02-12