qPCR – Phage Portal Primer Specificity Check

Stan Langevin recently identified a RLOv phage portal protein sequence that he wanted to see if this gene is incorporated in the withering syndrome bacteria (RLO) or the phage (RLOv). He designed a primer and probe set:

I’ve ordered/received the primers and need to test them out verify their specificity (via melt curve analysis) to ensure they only amplify a single target before proceeding with the expense of ordering a probe, as well as the time/effort that will be needed to, potentially, create a plasmid standard curve.

Set up qPCR on the following samples using the new RLOv phage portal primer set, as well as the RLOv DNA helicase qPCR assay to serve a positive control. These samples have been previously qPCR’d with the RLOv DNA helicase assay to establish the presence/quantities of RLOv in these samples. Samples are 1:1000 dilutions of the source DNA (made 20160106, but made fresh dilutions of RLOv negative samples today in H2O) due to the extremely high levels of RLOv detected in the RLOv positive samples.

SAMPLE RLOv +/-
08:4-3 NEGATIVE
08:4-4 NEGATIVE
08:4-15 POSITIVE
08:4-16 POSITIVE

 

RLOv phage portal master mix calcs (Google Sheet): 20160317 – qPCR XenoCal phage portal specificity

RLOv DNA Helicase master mix calcs (Google Sheet): 20160317 – qPCR RLOv

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Results:

qPCR Report (PDF): Sam_2016-03-17 08-03-13_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-17 08-03-13_CC009827.pcrd

The results are very odd and the qPCR should be repeated with fresh dilutions of the source DNA. Here are the reasons:

  • Almost no amplification with DNA helicase in RLOv+ samples
  • Amplification in all but one sample with phage portal – expected in all, none, RLOv- only, or RLOv+ only
  • Phage portal melt curves differ across samples – even within same sample type (i.e. RLOv-
  • Multiple peaks in phage portal melt curve in one RLOv- sample
  • Single peak in phage portal melt curve in the RLOv+ sample

See the amplification plots and melt curves below for a better idea of what’s happening.

 


qPCR Amplification Plots (DNA helicase in green; Phage portal gene in magenta)


 

 

qPCR Amplification Plots of Phage Portal Gene

 

qPCR Melt Curves of Phage Portal Gene

2 thoughts on “qPCR – Phage Portal Primer Specificity Check

  1. Pingback: qPCR – Repeat Phage Portal Primer Specificity Check | Sam's Notebook

  2. Pingback: qPCR – Repeat Phage Portal Primer Specificity Check | Sam's Notebook

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