Appendix B. Detailed molecular techniques used in prey DNA identification analyses.
Extraction of DNA from scats was carried out using the QIAamp DNA Stool Mini Kit (Qiagen). Samples were resuspended in the storage ethanol and then 1.5 mL of the ethanol/scat slurry was removed and centrifuged for 30 s at 4000 x g. The ethanol was poured off and the dry weight of the pellet was determined. All remaining steps followed the manufacturer’s instructions, except that only half the recommended volume of buffers/ InhibitEX tablets was used. The buffer volumes were cut down to reduce the risk of crossover contamination by minimizing the number of pipetting steps and by reducing the volume of liquid loaded into spin columns and tubes. The DNA was eluted in 100 μL Tris buffer (10 mM).
To minimize the probability of sample contamination (which increases when dealing with degraded DNA), all prey and soft scat DNA extractions and PCR reactions were carried out in a restricted (forensic) laboratory. Both extractions and PCR reactions were performed in separate Labconco Purifier PCR enclosures that were cleaned and UV treated between uses to ensure no cross contamination of genetic material occurred.
PCRs reactions were performed using 2 μL of template (prey, scat, or bone extracted DNA or primary PCR) in a total volume of 25 μL (primary PCRs) or 50 μL (nested PCRs) containing 20 μM of each primer, 0.2 mM dNTPs, 2.0 mM MgCl2, 1X BSA, 2X Qiagen Taq buffer, and 0.625 or 1.25 units of Hotstart Taq (Qiagen, Maryland, USA), respectively. All of the cycling conditions are noted in Table 2b.
The DCodeTM Universal Detection Mutation System (Bio-Rad, Hercules, California, USA) was used for DGGE. Polyacrylamide gels containing the appropriate % linear denaturant gradient (Table 2b) were formed with urea and formamide, and were buffered with 1X TAE. A total of 3 μL of PCR product (pooled or single PCR) was loaded into each well of the parallel gradient gel. After the appropriate running conditions were applied (Table 2b), gels were scanned with 505 nm (6-FAM) or 605 nm (ROX) band-pass filters using the default settings for acrylamide gels on the Hitachi FMBIO TM II fluorescent imaging system (MarioBio, Alameda, California, USA).
Sequencing reactions were carried out following the manufacturer’s instructions with BDT v3.1 (Applied Biosystems, Foster City, California, USA), purified using the DyeEX 2.0 spin kit (Qiagen, Maryland, USA) and electrophoresed on a 3730 Capillary DNA Sequencer (Applied Biosystems, Foster City, California, USA). Nucleotide sequences were edited and assembled with Sequencher 4.5 (Gene Codes Corp, Ann Arbor, Michigan, USA). Similarity matrices were calculated using the Jukes-Cantor method and Neighbour-joining dendrograms containing sequences of prey, scat items, and related species constructed in Mega 3.0 (Kumar et al. 1993) and visualized in TreeExplorer (ibid).