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Appendix A
PCR and Sequencing Protocols
We first optimized our PCR protocols and tested primer specificity of various mammal,
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reptile, bird, and insect DNA samples before running any mosquito samples. All PCRs were set
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up using PureTaq Ready-to-Go PCR beads (GE Biosciences) in 25 µl reactions using 1 mM of
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each primer and 2 µl of DNA extracted from mosquito abdomens. PCR conditions were an initial
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denaturation of 3 min at 94ºC, followed by 35 cycles of 94ºC for 30 sec, 52ºC for 20 sec, and
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72ºC for 1 min, with a final extension at 72ºC for 10 min. A positive control (2 μl template
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mammal DNA from a fresh tissue biopsy of a fruit bat, Pteropus hypomelanus) was included to
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ensure each PCR was successful. Negative controls (no DNA template added) were run in
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tandem with all other PCR reactions, with all reactions in the mix including ddH20. Products
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were visualized on a 1% agarose gel with CyberSafe (Invitrogen, Carlsbad, CA; 10 µl per 100 µl
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of gel), and positive amplifications were cleaned with the AMPure reagent (Agencourt, Beverly,
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Massachusetts) and sequenced in both directions using BigDye v. 3.1 (Applied Biosystems,
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Foster City, California) with the same primers that were used in amplification. Sequences were
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cleaned with CleanSeq (Agencourt, Beverly, Massachusetts) and run on an ABI 3730xl
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automated sequencer. Sequences were edited in Sequencher (GeneCodes, Madison, Wisconsin)
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and each was subjected to Megablast and BLASTn searches against all available sequences in
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GenBank.