qPCR – RLOv Specificity Check

After yesterday’s confirmation that the primer/probe sets for RLOv DNA helicase and head-to-tail were functional and specific for the RLOv (and don’t amplify RLO alone), I needed to confirm that the qPCRs only generated a single product in each reaction via melt curve analysis.

Primers tested:

  • RLOv_DNA_helicase
  • RLOv_head_to_tail_gene

Template DNA:

  • 06:6-54

NOTE: Remaining volume of template DNA wasn’t going to be sufficient for all reactions, so added 100μL of NanoPure H2O. Seeing how early the amplification was in yesterday’s qPCR (Cq ~15), this dilution should be fine.

All samples were run in duplicate.

Master mix calcs are here: 20151009 – qPCR RLOv

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:
qPCR Report (PDF): Sam_2015-10-09 12-36-54_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-10-09 12-36-54_CC009827.pcrd

Both primer sets amplified a single PCR product. This is demonstrated by the single melt peak for each primer set.

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