Site selection workflow and EMSAs on cloned fragments.

<p><b>A)</b> Outline of the selection procedure carried out to determine the DNA binding motif of Mid. In the first round, oligonucleotides consisting of a random 26 nucleotide core flanked by primer sequences were incubated with mid_Tbx. After purification and PCR amplification of co-precipitated fragments, the PCR amplified fragments were used in subsequent rounds of selection. <b>B)</b> All 54 oligonucleotide fragments were cloned and used as a template to create probes for EMSAs. Each probe was tested for recognition by Mid_Tbx. Probes positive for shifts, such as 2, 4, 57, 67 and 57 were tested a minimum of two times. Probes negative for shifts, such as 5, 10, 22, 48, and 64 were tested 3–5 times to exclude the possibility of false negatives. Arrows point to streaks often seen in probes that were weakly bound. Probes that did not display a noticeable, shifted band were considered to be unrecognized.