mt-genome of lasiorhinus krefftii: blood
We amplified the mtDNA genomes (excluding the control region) in three overlapping long-range fragments using primers developed by Mitchell et al. (2014) see SI for long-range PCR cycling conditions (SI).  Initially we attempted to amplify a fourth fragment that spanned the control region however it did not amplify consistently so was excluded. All three fragments were amplified in 25 L PCR reactions containing 5 L of 5x Expand Long Range Buffer 2 (including 12.5 mM MgCl2), 1.25 L of 10 M dNTPs, 0.75 L of each 10 M primer, 0.75 L of 100% DMSO, 0.35 L of Expand Long Range Polymerase, and 14.15 L of molecular grade water. Thermocycling involved 2 min at 92C initial denaturation, then 10 cycles of 92C for 10 s, 55C for 15 s, 68C for 7 min, then 25 cycles of 92C for 10 s, 55C for 15 s and 68C for 7 min plus 20 s per cycle. Final extension was 68C for 5 min.  We visualised PCR products on a 1% agarose gel to confirm amplification. Samples that did not amplify for any one of the three fragments were not carried forward into library preparation. After visualisation, we cleaned PCR products using a 0.6x volume of AxyPrep MAG PCR Clean-Up Kit (Axygen), followed by quantification with a Qubit 2.0 Broad Range DNA Kit (Invitrogen). We combined individual fragment PCR products in equimolar amounts before proceeding to library preparation using an Illumina Nextera XT DNA Library Preparation Kit and Indexes following the manufacturers instructions. Final libraries were visualised and quantified on an Agilent TapeStation 4150 using a D1000 ScreenTape.