qPCR – Withering Syndrome qPCR Assay Sample Checks

Ran qPCR on a set of water filter, fecal and tissue DNA extractions of varying copy number (based on Nate’s previous pinto abalone results) in order to get a set of high, medium and low copy number samples of each type for use in running the reproducibility aspect of the qPCR assay validation. Master mix calcs are here

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

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

Here’s the list of samples analyzed and a comparison with Nate’s original data. In all instances, my results show significantly lower copy numbers (orders of magnitude lower) than Nate’s. Whether this is due to sample degradation over time is unknown. All samples have been stored at -20C since ~2005. I will discuss with Lisa and select the samples that provide us with the best range of copy numbers for use in the assay validation.

qPCR – Immomix Lot Number Comparison

Here we go again. Testing out Sammi’s vial of replacement batch lot #111A vs. my vial vs. lot #110C. Master mix calcs are here. Using the p16RK3 3e6 standard curve sample (from 20120730) as a positive control as well as a red abalone DNA fecal extract (extracted by Lisa 9/12/11; labelled “Red 1 +”) that Sammi used in her successful PCR with the lot #111A. Plate layout, cycling params, etc can be found in the Results (see below).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All three master mixes amplified AND look absolutely identical!!! How is this even possible? Whatever. I’ll go ahead and run the second of three plates to assess analytical sensitivity for the withering syndrome qPCR assay validation.