Re-ran the qPCR (see earlier entry from today), due to the Low Feces samples failing to amplify. Replaced it with:
R3E 5/14/09 – 10^0
Everything else (including master mix, cycling params, etc) is all the same. See the earlier entry for details.
qPCR Data File (CFX96): Sam_2012-10-22 13-37-42_CC009827.pcrd
qPCR Report (PDF): Sam_2012-10-22 13-37-42_CC009827.pdf
Things looked good. The replacement Low Feces sample amplified.
Ran qPCR for the reproducibility aspect of the WSN qPCR Assay Validation. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the Results (see below).
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.
Samples used for “low”, “medium” and “high” copy numbers for each sample type are below, with expected fold copy number (based off of previous qPCRs):
Low: R4E 4/17/09 – 10^0
Med: R3E 7/23/09 – 10^3
High: R4E 7/23/09 – 10^4
Low: 09:16-18 – 10^1
Med: 09:16-22 – 10^2
High: 09:20-11 – 10^5
Low: 494:11-11 – 10^0
Med: 494-11-12 – 10^2
High: TAF SD A2 – 10^3
qPCR Data File (CFX96): Sam_2012-10-22 16-16-19_CC009827.pcrd
qPCR Report (PDF): Sam_2012-10-22 16-16-19_CC009827.pdf
Everything looked good except for the Low Feces sample which didn’t produce any amplification. Will identify another sample to use for the Low Feces sample.