Tag Archives: 08:13-18

Data Summary – Black Abalone Phage qPCRs

A quick summary table of the various black abalone qPCRs I ran yesterday:

SAMPLE RLO_MCP RLO_ph_protease XC_prophage_portal RLOv_DNA_helicase WSN
06:06-50  +  +  +  +  +
06:06-52  +  +  +  +  +
07:12-01  -  -  -  +  -
07:12-02  -  -  -  -  -
08:13-05  +  +  +  -  +
08:13-18  +  +  +  -  +
08:13-24  +  +  +  -  +*
08:13-25  +  +  +  -  +
  • This sample technically showed amplification, but came up after the last point on the standard curve. Most likely due to extremely low concentration (~0.5ng/uL).

  • RLO Major Capsid Protein (RLO_MCP)

  • RLO Prohead Protease Protein (RLO_ph_protease)
  • XenoCal Phage Portal Gene (XC prophage)
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qPCR – WSN on Black Abalone

Ran qPCRs on a set of black abalone digestive gland DNA (sample list provided by Carolyn):

07:12-01 (Black Ab exp 1)
07:12-02 (Black Ab exp 1)
08:13-05 (Black Ab exp 2)
08:13-18 (Black Ab exp 2)
08:13-24 (Black Ab exp 2)
08:13-25 (Black Ab exp 2)
UW06:06-32
UW06:06-41
UW06:06-50 (Black Ab exp 1)
UW06:06-52 (Black Ab exp 1)

The two samples with a strikethrough did not have any DNA left in the tubes and were not run.

All samples were run in duplicate.

Standard curve was p18RK7 from 20161128.

Cycling params, plate layout, etc can be seen in the qPCR Report (see Results).

Baseline was set 580 as previously determined by Lisa.

Results:
qPCR Report (PDF): Sam_2017-04-13%2016-20-54_CC009827_WSN1.pdf
qPCR Data File (CFX): Sam_2017-04-13%2016-20-54_CC009827_WSN1.pcrd

Standard curve looked good.

The following samples did not amplify:
– 07:12 set
– Note: 08:13-24 technically did amplify, but comes up below the lowest point of the standard curve, so technically it is effectively “no amplification”.

The remaining samples all came up positive.

Will convey to Carolyn and Stan.

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qPCR – RLOv DNA Helicase on Black Abalone

Ran qPCRs on a set of black abalone digestive gland DNA (sample list provided by Carolyn):

07:12-01 (Black Ab exp 1)
07:12-02 (Black Ab exp 1)
08:13-05 (Black Ab exp 2)
08:13-18 (Black Ab exp 2)
08:13-24 (Black Ab exp 2)
08:13-25 (Black Ab exp 2)
UW06:06-32
UW06:06-41

UW06:06-50 (Black Ab exp 1)
UW06:06-52 (Black Ab exp 1)

The two samples with a strikethrough did not have any DNA left in the tubes and were not run.

All samples were run in duplicate.

Standard curve was from 20161106.

Cycling params, plate layout, etc can be seen in the qPCR Report (see Results).

Baseline was set 580.5 as previously determined.

Results:
qPCR Report (PDF): Sam_2017-04-13%2016-20-54_CC009827_RLOv_helicase.pdf
qPCR Data File (CFX): Sam_2017-04-13%2016-20-54_CC009827_RLOv_helicase.pcrd

Standard curve looked good, although efficiency is pushing it on the high end.

The following samples did <em>not</em> amplify:

  • 07:12-02
  • All 08 samples.

The remaining samples all came up positive, with the 06 set being extremely hot (came up around cycle 13).

Will convey to Carolyn and Stan.

 

 

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qPCR – RLO Prophage Genes

Ran qPCRs on a set of black abalone digestive gland DNA (sample list provided by Carolyn):

07:12-01 (Black Ab exp 1)
07:12-02 (Black Ab exp 1)
08:13-05 (Black Ab exp 2)
08:13-18 (Black Ab exp 2)
08:13-24 (Black Ab exp 2)
08:13-25 (Black Ab exp 2)
UW06:06-32
UW06:06-41

UW06:06-50 (Black Ab exp 1)
UW06:06-52 (Black Ab exp 1)

The two samples with a strikethrough did not have any DNA left in the tubes and were not run.

Gene targets:
– RLO Major Capsid Protein (RLO_MCP)
– RLO Prohead Protease Protein (RLO_ph_protease)
– XenoCal Phage Portal Gene (XC prophage)

Master mix calcs are here (Google Sheet): 20170413 – qPCR_XCphagePortal_RLOcapsid_RLOprohead

The same master mix calculations were used for each, just swapped in appropriate primers.

All samples were run in duplicate.

Cycling params, plate layout, etc. can be found in the qPCR Report (see Results below).

Results:
qPCR Report (PDF): Sam_2017-04-13 14-56-03_CC009827.pdf
qPCR Data File (CFX): Sam_2017-04-13 14-56-03_CC009827.pcrd

Melt curves for all three primer sets looked perfect (see below)

Amplification present for all samples, with all three primers except the 07:12 samples.

Will pass info along to Carolyn and Stan.

Will add info to the following two spreadsheets (Google Sheets):

Black Abalone: Expt 1 – WS & Phage

Black Abalone: Expt 2 – WS only

 


 

Green = RLO_ph_protease

Brown = RLO_MCP

Blue = XC_prophage

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DNA Quantification – Black Abalone DNA (Black Ab Exp. 2)

Lisa recently isolated DNA from the following samples:

08:13-05 (Black Ab exp 2)
08:13-18 (Black Ab exp 2)
08:13-24 (Black Ab exp 2)
08:13-25 (Black Ab exp 2)

I quantified the samples using the Roberts Lab Qubit 3.0 with the Qubit ds High Sensitivity kit. Used 1uL of each sample.

Samples were stored in designated boxes in -20C in Rm. 240.

Results:

Qubit output (Google Sheet): 20170413_DNA_quantification_qubit

 

SAMPLE CONCENTRATION (ng/uL)
08:13-05 62.4
08:13-18 0.536
08:13-24 0.454
08:13-25 8.8

NOTE: The entirety of sample 08:13-24 will be provided to Stan Langevin for high-throughput sequencing.

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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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