Amplicon sequencing of Borrelia afzelii ospC: Myodes glareolus ear tissue : Sample 20081267
"DNA was extracted according to Laird et al. (1991) from ear skin biopsies (2 mm diameter) of Borrelia afzelii infected bank voles (N=50), yellow-necked mice (N=50) and common shrews (N=24). Partial B. afzelii ospC gene fragments (618bp) were amplified from all samples with fusionprimers containing primers ospC Fn and ospC Rn (Bunikis et al. 2004). In addition, 20 of the samples [bank voles (N=8), yellow-necked mice (N=6) and common shrews (N=6)] were technically replicated. Each PCR reaction (15 ml) contained 25 ng DNA-template, 0.2 mM of each fusionprimer (forward MID-tagged) and 1x Qiagen Multiplex PCR mastermix. The cycling conditions were: 95C for 15 min, 38 cycles of 95 for 30s, 52 C for 90s and 72 C for 60s, followed by an extension step at 72C for 10 min. Sequencing of amplicons was performed on the 454 Life Sciences Genome Sequencer FLX+ platform (Roche)."