Appendix D Terrestrial and Aquatic Biodiversity - Environment ...

SZ247: Bat survey from the Western Desert Resources Towns River project area, NT
mitochondrial DNA markers (bases 49–706 of the Cytochrome Oxidase I gene [COI]; bases
1–801 of the Cytochrome-b gene [Cytb]) were then amplified by PCR in 25 uL volumes
containing a final concentration of 1x PCR buffer (Applied Biosystems), 2 mM MgCl, 250 uM
of each dNTP, 5 uM each primer, 1 unit of Amplitaq Gold (Applied Biosystems) and 1 uL of
1/20 diluted template DNA. The following primers were used (names from the database of
the Evolutionary Biology Unit of the South Australian Museum): COI: forward M1243
(containing an M13f-21 tail; underlined)
5'-TGTAAAACGACGGCCAGTTCTCAACCAACCACAAAGACATTGG-3', reverse M1244
(containing an M13-pUCR tail; underlined)
5'-CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCAAAGAATCA-3' (VF1_t1 and
VR1_t1 of Ivanova et al. 2007); Cytb: forward M1226
5'-AATGACATGAAAAATCACCGTTGT-3' (Molcit-f of Ibáňez et al. 2006); reverse M040
5'-AAATAGGAARTATCAYTCTGGTTTRAT-3' (MVZ16 of Smith and Patton 1993). PCR
thermocycling conditions were as follows: initial denaturation at 94°C for 5 mins, followed by
35 cycles of 94°C for 30 secs, 50°C for 30 secs and 72°C for 1 min, with a final extension of
72°C for 10 mins. PCR products were purified on a vacuum manifold with a Multiscreen ® 384
PCR plate (Millipore), and the subsequent steps were outsourced to the Australian Genome
Research Facility. Products were sequenced using Big Dye Terminator ver. 3.1 chemistry
using the 'forward' primers only, and run on an ABI DNA capillary sequencer.
Sequences were edited and aligned manually in Bioedit ver. 7.09 (Hall 1999), and trimmed to
equal length for further analysis. A distance based Neighbour Joining phylogram was
created in PAUP* ver. 4.10b software (Swofford 2002) to illustrate the genetic distance
relationships amongst samples.
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