E4 breakpoint primers

YOR: 5'-ACCCTCGTTAGTTAGTCTTAG-3'
YGL: 5'-GCTCTCAAATTCGGCTGAAG-3'

E7 breakpoint primers

YOL: 5'-GTTGGCATTTCGGTCTTTACC-3'
YOR: 5'-ACAAGTCACCAATGACTGAC-3'

Both PCRs were set up as follows:
1 microliter 1:20 dilution appropriate DNA
5 microliters 10X Taq buffer
2 microliters 50 mM magnesium chloride
5 microliters 5 micromolar forward primer
5 microliters 5 micromolar reverse primer
5 microliters 2 mM dNTPs
0.5 microliters Taq
26.5 microliters water

PCR was performed on a gradient block PCR machine with a 2 minute extend time. Products were specific to the evolved strains at 65 degrees C melting temperature. These products were gel-purified, cloned into a TOPO-TA vector, and sequenced using M13 forward and reverse primers by the Stanford PAN facility.