that DNA extractions were successful, aliquots of extracted DNA (e.
g., 8 µL) are mixed with a few microliters of agarose loading dye,
investigated via 2% agarose gel electrophoresis (185 V, 20 minutes
in 1XTBE buffer), and documented using a UV light transilluminator. When 2% agarose gels are prepared, 4 µL of SYBR Safe gel stain
are added to 100 mL of molten agarose before the gel is poured.
Once the successful extraction of DNA is confirmed, a multiplex
PCR (25 µL reaction) is prepared to target the pathogen. The multiplex PCR in this exercise uses two primer pairs: one based on the 18S
ribosomal gene to target fungi in general (RDS478/RDS482, ~350 bp),
and one Coccidioides specific primer pair (ITSC1A/ITSC2, 220 bp) that
selectively amplifies a highly variable area of the ITS2 region of the
ribosomal gene (Greene et al., 2000; Lauer et al., 2012). The Green
GoTaq Mastermix is used to prepare 25 µL PCRs following recommendations of aliquots of DNA, primer, and sterile PCR water in the
manufacturer’s protocol. The success of the multiplex PCRs can be
investigated via 2% agarose gel electrophoresis viewed on a UV light
transilluminator. Details about PCR procedures, including cycling
Figure 2. Flowchart showing, step by step, how valley fever incidence data and soil parameter information can be obtained
from publicly available online sources. Students can use the data to further explore disease incidence over time by making a line
graph or by investigating differences of soil parameters for Coccidioides positive versus negative sites (Microsoft Excel also provides
tools to perform correlations between sets of values).