qPCR – Withering Syndrome Probes Comparison

Due to poor performance seen in yesterday’s second run, which used the IDT probe instead of the ABI probe, I will run the p18RK7 curve (from 20120731) with both the IDT and the ABI RLP_p probes to see if the probe is the underlying issue.

Master mix calcs are here: 20140827 – qPCR IDT-ABI Probe Comparison

All samples were run in duplicate.

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

Baseline threshold set to 580 RFUs.

Results:

qPCR Report (PDF): Sam_2014-08-27 10-32-19_CC009827.pdf

qPCR Date File (CFX96): Sam_2014-08-27 10-32-19_CC009827.pcrd

Quick summary: IDT probe is bad, ABI probe is good.

However, I’ve just realized that there might be more to this than just a bad probe from IDT. The amplification profile is suspiciously similar to what we were seeing with the bad probe(s) from Biosearch Technologies. The only thing that differentiates the IDT and Biosearch Technologies probes from the ABI probe is that the ABI probe already arrived reconstituted at 100uM. Biosearch Technologies and IDT probes arrive lyophilized and have to be reconstituted by us. Is it possible that the buffer we’re using is degrading the probes? Will order new IDT probe and order IDT’s version of low TE buffer (called IDTE).

Here are the amplification and standard curve plots:

IDT Amplification

IDT Standard Curve

ABI Amplification

ABI Standard Curve

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