A 20-μmol amount of Linker1 and a 20-μmol amount of Linker2 (see later description) were mixed, denatured for 5 min at 95°C, incubated at 70°C, slowly cooled to room temperature, and stored overnight at 4°C. For first-strand synthesis, 2 μg of piperidine-digested DNA was mixed with 5× Herculase II reaction buffer (Agilent Technologies Deutschland GmbH, Böblingen, Germany), 0.625 μl of dNTP (10 μM), 0.5 μl of genomic primer (1 μM), and 0.2 μl of Herculase II and filled up to 30 μl with PCR-grade water, with subsequent PCR (5 min, 95°C; 30 min, 60°C; 10 min, 72°C).
To ligate the linker, 10 μl of T4 DNA-Ligase Buffer (New England Biolabs), 10 μl of 50% PEG4000 solution (New England Biolabs), 5 μl of linker mix, and 2 U of T4 Ligase (New England Biolabs) were mixed with the PCR, added to 50-μl total volume with water, and incubated overnight at 4°C. DNA was precipitated and the pellet resuspended in 10 μl of 10 mM Tris-HCl + 0.1 mM EDTA.
To amplify the first strand, the following components were added in the following order: 36.85 μl of water, 10 μl of 5× Herculase II reaction buffer, 1.25 μl of dNTPs (10 mM), 0.2 μl of linker primer (100 μM; see later description), 0.2 μl of genomic primer (100 μM), 1 μl of ligation reaction, and 0.5 μl of Herculase II. To fluorescently label PCR products, 5 μl of ATTO-488 UTP (1 mM; Jena Bioscience, Jena, Germany) was added to the PCR.
To ligate the linker, 10 μl of T4 DNA-Ligase Buffer (New England Biolabs), 10 μl of 50% PEG4000 solution (New England Biolabs), 5 μl of linker mix, and 2 U of T4 Ligase (New England Biolabs) were mixed with the PCR, added to 50-μl total volume with water, and incubated overnight at 4°C. DNA was precipitated and the pellet resuspended in 10 μl of 10 mM Tris-HCl + 0.1 mM EDTA.
To amplify the first strand, the following components were added in the following order: 36.85 μl of water, 10 μl of 5× Herculase II reaction buffer, 1.25 μl of dNTPs (10 mM), 0.2 μl of linker primer (100 μM; see later description), 0.2 μl of genomic primer (100 μM), 1 μl of ligation reaction, and 0.5 μl of Herculase II. To fluorescently label PCR products, 5 μl of ATTO-488 UTP (1 mM; Jena Bioscience, Jena, Germany) was added to the PCR.