EvaGreen® Dye

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.	PCR plates, tubes and pipette tips should be UV sterilized for 20-30 mins

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
PCR plates, tubes and pipette tips should be UV sterilized for 20-30 mins

Publication protocol

PCR assays for detecting fungi
To detect fungi, the LightCycler Nano (Roche Applied Science) and PCR-clean tubes were used as described above. During the PCR, the PCR reaction mixture (20 μL) contained 2 μL of DNA template or 2 μL (8.0 ng/μL) of DNA extracted from Candida albicans as a positive control, or distilled water (NAKALAI TESQUE, INC.) as a negative control in 50 mM KCl, 2.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 200 μM of each deoxynucleoside triphosphate (dNTP), 0.25 μM each of Fungal universal primer, 1×EvaGreen (Biotium Inc.), and 2.0 units (0.4 μL) of conventional thermostable DNA polymerase (r-Taq: Toyobo, Osaka, Japan) supplemented with stock buffer solution. Each sample was incubated for three minutes at 95°C, then denatured for 10 seconds at 95°C, annealed for 15 seconds at 57°C, extended for 20 seconds at 72°C and subjected to fluorescence acquisition for two seconds at 82°C for 40 cycles.

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Manufacturer protocol

Download the product protocol from Biotium for EvaGreen® Dye below.

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Videos

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