How and Why Overcome the Impediments to Resolution: Lessons from rhinolophid and hipposiderid Bats

Foley, Nicole M.; Thong, Vu Dinh; Soisook, Pipat; Goodman, Steven M.; Armstrong, Kyle N.; Jacobs, David S.; Puechmaille, Sébastien J.; Teeling, Emma C.

Tissue Sampling, DNA Extraction, and Amplification Tissues used for genetic analyses were either from wing punches from released individuals or tissue samples from voucher specimens housed in different museum collections (see Genbank Accessions for further details). DNA was isolated using a modified salt/chloroform extraction protocol (Miller et al. 1988) , which included an additional chloroform/isoamyl alcohol (24/1) step after the addition of the saturated NaCl solution . Nuclear gene fragments, comprising exon and intron sequences of varying length were amplified with primers listed in supplementary table S1, Supplementary Material online. Reactions were carried out in 25 ml solutions containing 2 ml of DNA extract, 1X polymerase chain reaction (PCR) buffer minus Mg (Invitrogen), 1.5 mM MgCl 2 , 0.4 mM each primer, 0.2 mM dNTPs, and 1 U Platinum Taq DNA Polymerase High Fidelity (Invitrogen). Amplifications were carried out in a DNA Engine DYAD thermocycler (MJ Research) with PCR programs: 1) Touchdown 50-initial step 10' at 95 C, then ten cycles of 15" at 95 C, 30" at 60 C (with a reduction of 2 C every 2 cycles), 1' at 72 C, following by 30 cycles of 30" at 95 C, 30" at 50 C, and 1' at 72 C and a final step of 5' at 72 C and 2) touchdown 55-initial step 10' at 95 C, then ten cycles of 15" at 95 C, 30" at 65 C (with a reduction of 2 C every 2 cycles), 1' at 72 C, following by 30 cycles of 30" at 95 C, 30" at 55 C, and 1' at 72 C and a final step of 5' at 72 C.