Diversidad y control biológico de insectos - CyberTesis UACh ...

centrifugation and supernatant was discarded, 1 mL ethanol wash buffer was added, glass
powder was suspend with a pipet tip and glass powder was centrifugated again with a brief
centrifugation. Ethanol was discarded and tubes were dried on a heat block (55 ºC, 10 min).
Glass powder was suspended in sterile distilled water (100 µL) and heated for 1 min (55
ºC). Finally, glass powder was packed and eluted DNA was transferred to a clean tube and
stored at -20º C. Aliquots were used to check DNA quantity and quality.
Amplification and sequencing.
DNA sequences from the B fragment were obtained by cycle sequencing of PCR-amplified
DNA. PCR reactions were performed in 50 µL final volume containing 2 µL template
DNA, 5 µL of 10X PCR buffer (10 mM Tris/HCl pH 8.0, 50 mM KCl, 1.5-2.0 mM
MgCl2), 4 µL of dNTP mix (1.25 mM each dATP, dCTP, dGTP, and dTTP), 10 pmol each
of the opposing amplification primers, 0.5 µL Taq polymerase (Promega, Madison, WI).
The reactions were run in a MJ Research PTC-200 thermocycler, using a touchdown PCR
procedure (Rehner and Buckley, 2005). Primers B5.1 and B3.1R were used for spanning B
fragment (Table 3).
Before to separate the PCR products by electrophoresis, the PCR volumes were reduced in
a Speed-Vac (2 h, highest desiccation rate). Remnant volume was eliminated with a brief
spin. DNA was resuspended in 1X low EDTA (0.1 mM) TAE electrophoresis buffer to
which has been added 1/10 volume loading buffer. PCR products were loaded onto 1.5%
NuSieve agarose gels (Bio-Whitakker, Rockland, Maine), stained with ethidium bromide.
After the run, the gel was cooled (15 min, 5 ºC). The amplicons were visualized by
exposing the gel to UV light and excised using a broad-nosed scalpel blade. Gel slices were
frozen (-20 ºC) until sequencing. At this point, gel slices were thawed and centrifuged in a
microcentrifuge (20000 x g, 10 min) to compress the gel slice and extrudes DNA.
The sequencing reactions were performed with ABI BigDye 2.0 (Applied Systems, Foster
City, CA) with a total volume of 5 µl, which included 0.5 µl BigDye diluted in 1,5 µl
dilution buffer, 3 pmol primer, 100 ng gel-purified PCR template. Cycle sequencing was
performed in 96-well microtiter plates according to the manufacturer’s instructions. The
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