qPCR – Withering Syndrome NcoI Linearized Clones from 20120726

Performed a dilution series to have the desired copy number using Low TE Buffer. The calculations were performed using a spreadsheet developed by Nate. The calculations are here and the workbook has a separate sheet for each clone. qPCR master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Reports below (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report – p16RK3 standards (PDF)

qPCR Report – p16RK7 standards (PDF)

qPCR Report – p18RK7 standards (PDF)

SUCCESS!!! It’s a friggin’ miracle! Finally, we have three clones (pWC8 did not amplify at all!) that are useable for the withering syndrome assay!! Here are the r^2 values and the efficiency for each of the three clones that did work:

p16RK3 – r^2 = 0.996, E = 91.7%

p16RK7 – r^2 = 0.995, E = 98.4%

p18RK7 – r^2 = 0.998, E = 94.1%

Due to it having the best efficiency, p16RK7 clone will be used for the assay validation.

UPDATED 20121024 – Modified data file (and subsequently the qPCR Report) to have a baseline threshold of 400 and cycles to analyze 41 to match existing conditions used for the withering syndrome qPCR assay validation. HOWEVER, I have NOT updated the r^2 and efficiency numbers listed above to reflect the new baseline setting! Please see the respective qPCR Reports for that information!!

3 thoughts on “qPCR – Withering Syndrome NcoI Linearized Clones from 20120726

  1. Pingback: Samples for Abalone Withering Syndrome ddPCR | Sam's Notebook

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  3. Pingback: qPCR – Withering Syncrome ddPCR Comparison | Sam's Notebook

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