M13 reverse primer

Purified DNA was cloned using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Transformation was done using competent E. coli TOP10 cells provided by the manufacturer. The transformed cells were plated onto Luria-Bertani agar plates supplemented with kanamycin (50 µg/ml) and incubated overnight at 37°C. Each white colony was placed into a tube containing 40 µL of 10 mM Tris-HCl pH 8.0. One µL of the cell suspension was used directly as template for PCR with an M13 (−21) forward primer and an M13 reverse primer (Invitrogen). The amplicon was checked for correct size by electrophoresis on a 1% agarose gel. DNA from clones was purified to remove primer and dNTPs by treatment with exonuclease and shrimp alkaline phosphatase (Amersham, Pittsburg, PA).
Clones were screened by sequencing with primer Y31, 5’-TKACCGCGGCTGCTG-3’ (519–533 reverse). If the clone sequence differed by more than 7 bases from previously identified feline oral reference sequences, the remaining amplicon DNA was purified using QIAquick PCR purification kits (Qiagen, Valencia, CA) and fully sequenced with additional primers (Dewhirst et al., 2012 (link)).