Tag Archives: 06:6-54

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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Data Aggregation – WS RLO and RLOv DNA Helicase qPCR and WS RLO Infection Intensities

Carolyn asked me to send her the data described above.

RLOv DNA helicase qPCR data were grabbed from the qPCR I ran on 20160106.

The qPCR data for withering syndrome RLO were culled from these three different spreadsheets:

 

The summary is below. I have emailed a copy of the spreadsheet to Carolyn.

Google Sheet: 20160404_Summary_RLO_RLOvDNAhelicase_qPCR_HistoIntensities

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qPCR – Black Abalone DNA with Varying Levels of RLO/RLOv

Carolyn & Stan Langevin have agreed that the DNA helicase qPCR should be tested on 10 black abalone DNA extractions that fall into multiple levels of the Friedman Lab’s withering syndrome histology scoring.

Earlier today I identified samples at three different histology scoring levels of RLO: 0, 1, & 2.

Here’s the list of samples that will be qPCR’d. There were only eight samples that had histology scores of 2 in both PE and Dg.

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
06:5-18 06:5-35 06:5-31
06:5-30 06:6-32 06:5-32B
06:50-04 06:6-39 06:6-46
06:50-05 06:6-42 06:6-49
07:12-01 06:6-44 08:3-05
07:12-02 06:6-52 08:3-07
07:12-03 06:6-54 08:3-15
07:12-04 06:50-08 08:3-16
07:12-07 06:50-10
07:12-09 07:12-18

 

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate on the CFX96 (BioRad).

Master mix calcs are here: 20151120 – qPCR RLOv Black Abs

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

Results:
qPCR Report (PDF): Sam_2015-11-20 15-00-27_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-11-20 15-00-27_CC009827.pcrd

Quick summary of the results:

  • 50% of the RLO/RLOv 0 score samples are positive for RLOv DNA helicase. Will talk to Carolyn to see if she has withering syndrome qPCR data for these samples to compare RLOv-positive samples with WSN-positive samples. If not, will run withering syndrome qPCR.
  • All RLO/RLOv 1 & 2 scored samples are positive for RLOv DNA helicase
  • All RLO/RLOv 2 scored samples come up before the standar curve; these should be diluted and re-run.
  • Standard curve isn’t perfect (the 3 copy sample is throwing it off).

 

STANDARD CURVE AMP & SCATTER PLOTS

 

 

RLO/RLOv 0 AMP PLOTS

 

 

RLO/RLOv 1 AMP PLOTS

 

 

RLO/RLOv 2 AMP PLOTS

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PCR – RLOv for Cloning & Sequencing

After yesterday’s confirmation that the qPCR primer/probe sets for RLOv DNA helicase and head-to-tail were functional and specific for the RLOv, I needed to generate PCR products to clone and sequence.

Primers tested:

  • RLOv_DNA_helicase
  • RLOv_head_to_tail_gene

Template DNA:

  • 06:6-54

All samples were run in duplicate.

Master mix calcs are here: 20151009 – PCR RLOv

Cycling Params (PTC-200; MJ Research)

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 95
  • 600
40 Cycles
  • 95
  • 55
  • 72
  • 15
  • 15
  • 30

Samples were run on a 0.8% agarose 1x TBE gel, stained with ethidium bromide.

Results:

Amplification looks great. No amplification in no template controls (NTCs). Excised bands and purified products using Ultrafree DA Spin Columns (Millipore). Samples will be stored @ 4C until I am able to clone them for sequencing.

 

Gel image showing excised bands. And, it’s a complete hack job, which is embarrassing…

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qPCR – RLOv Specificity Check

After yesterday’s confirmation that the primer/probe sets for RLOv DNA helicase and head-to-tail were functional and specific for the RLOv (and don’t amplify RLO alone), I needed to confirm that the qPCRs only generated a single product in each reaction via melt curve analysis.

Primers tested:

  • RLOv_DNA_helicase
  • RLOv_head_to_tail_gene

Template DNA:

  • 06:6-54

NOTE: Remaining volume of template DNA wasn’t going to be sufficient for all reactions, so added 100μL of NanoPure H2O. Seeing how early the amplification was in yesterday’s qPCR (Cq ~15), this dilution should be fine.

All samples were run in duplicate.

Master mix calcs are here: 20151009 – qPCR RLOv

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

Results:
qPCR Report (PDF): Sam_2015-10-09 12-36-54_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-10-09 12-36-54_CC009827.pcrd

Both primer sets amplified a single PCR product. This is demonstrated by the single melt peak for each primer set.

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qPCR – New Withering Syndrome Phage Primers

Ran qPCR with the newly designed primers and probes for the following targets:

  • DNA Helicase (RLOv)
  • Head-to-tail gene (RLOv)
  • WSN1 (RLO)

Template DNA used:

In the histology scoring pictures below, the “New” column refers to histology scores for the presence of the phage. A score = 0 means no phage.

  • 06:5-6 (RLO only)

  • 06:6-54 (RLOv)

  • UW08:22-11A (naive pinto abalone; no RLO)

 

Master mix calcs are here: 20151008 – qPCR WS phage

All samples were run in duplicate. Cycling params, plate layout, etc. can be seen in the qPCR Report (see below).

Results:

qPCR Report (PDF): Sam_2015-10-08 17-45-38_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-10-08 17-45-38_CC009827.pcrd

ORANGE – WSN1; BLUE – DNA Helicase; GREEN – Head-to-tail

The results look great! The two RLOv (phage) primer sets only amplify in the sample that has histological confirmation of the presence of phage (06:6-54). They do not amplify in the RLO-only (no phage; 06:5-6) sample, demonstrating that these two primer sets are indeed specific to the phage and don’t  amplify the RLO.

The withering syndrome primers (WSN1) were run to confirm that there aredetectable levels of RLO in both the RLOv & RLO samples, to further support the evidence showing the specificity of the two phage primer sets.

Will use the two RLOv primer sets in a conventional PCR for cloning/sequencing and development and validation of a qPCR standard curve.

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