My colleagues and I loaded two gels with a 200 bp DNA ladder, a blank (negative control), two amplified Ranavirus samples (positive controls), and amplified samples of gDNA from tail tips from frogs, toads and salamanders using MCP-1F and MCP-6R primers.  The results in the attachment were suggested the presence of Ranavirus for the two positive controls but the primers did not result in amplicons for the negative control, or for the frog and toad samples.  However, bright bands appeared in the lanes for the four newt samples (N. viridescens) but the size of the bands was only about 400 bp.  Upon sequencing, the newt amplicons exhibited lots of “noise” and did not give any matches when blasted in GenBank.  Thinking the result might be linked to a low annealing temperature (50C for 2 minutes), we raised the annealing temperature to 55C.  Oddly, the salamander bands were bright as ever and the positive samples a bit lighter!  Has anyone else experienced this?  Any explanations would be helpful!  And thank you for considering the question.

Terry Schwaner, Ph.D.

Dean, Center for Life & Health Sciences
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