Tag Archives: 08:13-25

qPCR – Black Abalone with XC Prophage Portal Primers

Ran qPCR with black abalone samples from the 1st and 2nd experiments to see if the Xenocal prophage portal gene is detected.

Master mix calcs (Google Sheet): 20160421 – qPCR Black Abs XenoCal phage portal

All samples were run in duplicate.

Black abalone sample 08:13-2 was run as a positive control.

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

Results:
qPCR Report (PDF): Sam_2016-04-21 14-11-09_CC009827.pdf
qPCR Data File (CFX): Sam_2016-04-21 14-11-09_CC009827.pcrd

Two samples failed to produce amplification: 06:6-44 and 07:12-18. All other samples amplified. Will compile this data with WSN and RLOv DNA helicase and send along to Carolyn and Stan.

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qPCR – WSN1 & RLOv DNA helicase on Black Abalone 2nd Experiment 08:13 Accessions

Checking DNA isolated earlier today from the 2nd black abalone experiment to see if withering syndrome (RLO) and/or the withering syndrome phage (RLOv) is detectable in these samples.

Master mix calcs

Standard curves

All samples were run in duplicate.

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

Baseline thresholds were set to the following values for each assay (RLOv threshold determined by me on 20160128; WSN1 threshold determined by Lisa):

RLOv DNA helicase: 580.5

WSN1: 580

Results:

qPCR Report – RLOv DNA helicase (PDF): Sam_2016-04-21 12-39-33_CC009827_RLOv_DNA_helicase.pdf
qPCR Report – WSN1 (PDF): Sam_2016-04-21 12-39-33_CC009827_WSN.pdf
qPCR Data File (CFX): Sam_2016-04-21 12-39-33_CC009827.pcrd

RLOv DNA helicase does not amplify in any samples.

WSN1 amplifies in all samples.

All samples are RLO+/RLOv-, as seen in the previous set of 08:13 samples that I qPCR’d.

 

RLOv DNA Helicase Standard Curve

 

 

RLOv DNA Helicase Amplification (Green = Std Cuve, Blue = Samples)

 

 

 

WSN1 Standard Curve

 

WSN1 Amplification (Blue = Standard Curve, Magenta= Samples)

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DNA Isolation – Black Abalone 2nd Experiment 08:13 Accessions

Isolated DNA from EtOH-preserved black abalone digestive gland tissue from the 2nd black abalone experiment.

There’s some odd background in regards to these samples which I previously described here that might be worth reviewing.

DNA was isolated using the QIAamp Fast DNA Stool Kit (Qiagen). Tissues were weighed and briefly homogenized with a disposable pestle in InhibitEX Buffer. Manufacturer’s protocol was followed. DNA  was eluted in 100μL of Buffer ATE and quantified on the Roberts Lab Qubit3.0 (ThermoFisher) using 1μL with the Qubit dsDNA Broad Range assay.

Results:

Google Sheet: 20160421_DNA_isolation_08:13_subset

 

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