M13 reverse

In order to determine the full-length genome of the Italian DogCV prototype strain, a rolling circle amplification (RCA) protocol was performed as previously described [31] (link) using the TempliPhi 100 amplification kit (Amersham Biosciences). Briefly, 1 µl of extracted DNA was mixed with 5 µl of TempliPhi sample buffer supplemented with 450 µM dNTPs, and the mix was incubated at 95 °C for 3 min and subsequently cooled on ice. After adding 5 µl TempliPhi reaction buffer and 0.2 µl TempliPhi enzyme mix, the mixture was incubated at 30 °C for 16 h, and subsequently inactivated at 65 °C for 10 min. For cloning, a total of 30 µl of the RCA product was digested with ApaI and the resulting 2 kb-long fragment was ligated with the ApaI-restricted vector pBluescript II SK(+) (Stratagene) and transformed into XL-1 Blue MRF' Escherichia coli cells (Stratagene). The 2 kb-long insert of the plasmid was sequenced using the universal primers M13 forward and M13 reverse (Invitrogen) at the BaseClear B.V. (Leiden, The Netherlands). A consensus sequence was generated using three clones.