DNA Isolation and Analysis
We extract DNA from the
cotton swabs using MoBio's PowerSoil™ DNA Isolation
kit and stored the eluted DNA samples at -20°C. In hopes
to develop a cost-effective and more effective procedure
to quantify Bd, we are using traditional PCR to work on detecting
the presence of amphibian DNA before any downstream applications.
By determining if we isolated amphibian DNA, which should
be present since swabbing dislodges epithelial cells,
we can rule out one category of false negatives during
Bd analysis. That category is Bd false negatives due to lack
of DNA in the sample. Amphibian screening samples are run
using a MJ Mini™ Gradient
Thermal Cycler. We use primers developed by Kocher et al.
1989 that amplify a specific region of ribosomal DNA
found in all amphibians (For more on primers, read Kocher
et al. 1989). Samples that test positive for amphibian
DNA are ready to test for the presence of Bd.
Inside of a MJ Mini™ (Top) and a autoradiogram of one of our gels (Bottom)
Photo
credits can be found on acknowlegements page
This site was designed and created by G. Bomar as part of a summer undergraduate
research program at Davidson College.
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