qPCR – Withering Syndrome Assay with New IDT Primers

It dawned on me that the qPCRs I ran on 20140909 comparing different stocks of primers, using the new IDT probe (which all ended up looking bad), all used primer stocks that had been reconstituted with low TE buffer made in the lab, as opposed to “store bought.” Could this actually be the reason that the IDT probes (which come lyophilized and require reconstitution prior to use) don’t seem to work, while the ABI probe (which comes as a liquid, 100uM stock) does?

Received new IDT primer stocks of WSN1F/R and reconstituted them with the store bought low TE buffer from IDT.

All samples were run in duplicate. See the qPCR Report (in Results below) for plate layout, cycling params, etc.

Baseline Threshold set to 580 RFUs, as previously determined by Lisa for use with the Promega master mix.

Results:

qPCR Report (PDF): Sam_2014-09-19 10-55-33_CC009827.pdf

qPCR Data File (CFX96): Sam_2014-09-19 10-55-33_CC009827.pcrd

I’ll be damned! Our lab-made low TE buffer seems to have been the culprit! Both IDT and ABI probes performed perfectly and are virtually identical. See the amplification plots and standard curve plots below for each of the two probes. Both exhibit R^2 values of 0.999.

ABI Probe Amplificaton

ABI Probe Standard Curve

IDT Probe Amplification

IDT Probe Standard Curve

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