Tag Archives: 08:13-16

qPCR – Black Abalone with XC Prophage Portal Primers

I accidentally skipped two samples from the 2nd black abalone experiment sample set that I qPCR’d last week, so I’m qPCRing them today.

Master mix calcs (Google Sheet): 20160425 – qPCR Black Abs XenoCal phage portal

All samples were run in duplicate.

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

Results:
qPCR Report (PDF): Sam_2016-04-25 12-55-40_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-04-25 12-55-40_CC009827.pcrd

Have amplification in both samples.

I will add this to a “master” spreadsheet that I’ve made containing qPCR data from three genes on ~20 samples from both the 1st and 2nd black abalone experiments.

 

 

 

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

Ran WSN1 and RLOv DNA helicase qPCRs on the black abalone DNA I extracted yesterday to assess whether or not these samples are RLO+/- and RLOv+/-. According to Carolyn (and this spreadsheet), they should all be RLO+/RLOv-, which is what I need in order to proceed with testing samples with the XenoCal prophage portal primers.

WSN1 Master Mix Calcs (Google Sheet): 20150330 – qPCR Black Ab 08:13 WSN1 Check

RLOv DNA Helicase Master Mix Calcs (Google Sheet): 20160330 – qPCR Black Ab 08:13 RLOv check

All samples were run in duplicate.

Plate layout, cycling params, etc. are in the qPCR Report (see Results section below).

RLOv DNA helicase standard curve from 20151224.

WSN p18RK7 standard curve from 20160316.

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 (PDF): Sam_2016-03-30 10-00-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-03-30 10-00-07_CC009827.pcrd

All samples are RLO+/RLOv-. This is great and can proceed with checking them with the XenoCal prophage portal primers.

 

RLOv DNA Helicase Standard Curve

 

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

 

 

WSN1 Standard Curve

 

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

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

Oddly, I was unable to find any DNA for the 08:13 samples that should have been previously qPCR’d for RLO.

Instead, I tracked down the EtOH-preserved digestive gland (DG) tissues from when these were initially sampled. The box contained both of the “QPCR” tissue samples, however, many of them had dried out. This fact had already been denoted on the outside of the box and on the tubes.

Finding these samples is a bit strange. It’s odd because if someone had performed qPCR analysis on these 08:13 samples, the DNA should’ve come from either of the two “QPCR” tissue samples; but, looking at the vials, it seems like no tissue has been removed from any of the tubes…

Additionally, despite the fact that the spreadsheet Carolyn provided me with the other day indicating that the 08:13 samples are from the 2nd black abalone experiment, the label on this box indicates that these are from the 1st black abalone experiment… Despite this, I’m fairly certain these are indeed from Experiment 2, as these accession numbers have never been brought up before in any of Lisa’s extensive work on the 1st black abalone experiment.

I extracted DNA using the QIAmp Fast DNA Stool Mini Kit (Qiagen) from the following samples. DNA was eluted with 100μL of Buffer ATE and quantified on the Roberts Lab Qubit3.0 (ThermoFisher) using 1μL.

ACCESSION
08:13-2
08:13-3
08:13-4
08:13-5
08:13-6
08:13-7
08:13-11
08:13-12
08:13-13
08:13-14
08:13-16
08:13-17

Results:

Google Sheet: 20160329_DNA_isolation_08:13_subset

Will run qPCRs (WSN1, RLOv DNA helicase, and XenoCal prophage portal) on these samples tomorrow.

DNA has been stored in an existing box in the full-sized -20C freezer in FSH240 and the label on the box has been updated to include these samples.

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