Tag Archives: WSN1

qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs were re-used from previous standard curve check (Google Sheet): 20161128 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2017-07-03 09-34-31_CC009827.pdf
qPCR Data File (CFX): Sam_2017-07-03 09-34-31_CC009827.pcrd

Curve looks good! Will use this one going forward. Will store in the withering syndrome standard curve box in the FSH 240 4C.

 

ALL MIXES

 

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qPCR – Check New Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

This is a qPCR to test the fresh p18RK7 dilution I made earlier today, and verify it works well (i.e. linear spread, good R^2 value, same Cqs as previous curve dilution, etc.).

Master mix calcs were re-used from previous standard curve check (Google Sheet): 20161128 – qPCR WS p18RK7 Curve Check

I made three separate master mixes to check the curve three times.

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Report (see Results below).

Baseline threshold was manually set to 580, as previously determined by Lisa for this assay.

Results:

qPCR Report (PDF): Sam_2017-06-29 12-49-46_CC009827.pdf
qPCR Data File (CFX): Sam_2017-06-29 12-49-46_CC009827.pcrd

 

I screwed something up in the dilution series. Seems difficult to screw up something so basic, but the results don’t lie. Will discard this curve and will remake the curve and re-test. Argh!

 

 

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qPCR – Ava’s RLO Transmission Samples

Due to a wonky standard curve, I repeated the qPCR from earlier this week.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170619_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

qPCR Report (PDF): Sam_2017-06-21 07-39-31_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-06-21 07-39-31_CC009827.pcrd

 

Well, unfortunately, it looks like the curve has gone wonky. This is two consecutive runs with the same behavior. A new curve will have to be made and tested.

 

 

 

 

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qPCR – Ava’s RLO Transmission Samples

Ran qPCRs on samples extracted earlier today. Also re-ran samples 15:08-29 and 15:09-145, per Ava’s request.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170619_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

qPCR Report (PDF): Sam_2017-06-19 13-10-22_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-06-19 13-10-22_CC009827.pcrd

 

These will need to be re-run, as the standard curve is a bit wonky (see below). Will re-run later this week.

 

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qPCR – Ava’s RLO Transmission Samples

Ran qPCRs on the DNA I extracted on 20170504 and earlier today.

The full list of samples is here (Google Sheet): 20170502_Ava_Ab_List

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170509_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

Curves look good on all runs (except the one that’s been noted and has been repeated). Will pass along to Ava and Carolyn.

qPCR Report (PDF): Sam_2017-05-09 07-29-36_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 07-29-36_CC009827.pcrd

 


This plate has a bad curve and needs to be re-run! It has been repeated below!

I’ve included this for posterity only!

qPCR Report (PDF): Sam_2017-05-09 08-56-22_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 08-56-22_CC009827.pcrd


 

 

qPCR Report (PDF): Sam_2017-05-09 10-21-15_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 10-21-15_CC009827.pcrd

qPCR Report (PDF): Sam_2017-05-09 11-44-42_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 11-44-42_CC009827.pcrd

 

qPCR Report (PDF): Sam_2017-05-09 13-07-46_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 13-07-46_CC009827.pcrd

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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 – CDFW White Abalone Samples (WSN1)

The samples that CDFW sent us earlier are intended for checking for the presence of the RLOv (phage), but I figured it would be prudent to verify that they were positive for the RLO as well. I ran these samples concurrently with some other samples I had to test with the withering syndrome qPCR assay.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here; since I ran these with the other samples, the master mix used was part of the other project indicated in the spreadsheet (Google Sheet): 20170406_qPCR_WSN1_capstone

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

Baseline threshold was manually set to 580, as previously determined by Lisa.

Results:

Standard curve looks good and all samples provided come up positive for RLO.

qPCR Report (PDF): Sam_2017-04-06%2011-36-53_CC009827_CDFW_white_ab_WSN1.pdf
qPCR Data File (CFX): Sam_2017-04-06%2011-36-53_CC009827_CDFW_white_ab_WSN1.pcrd

 

Amplication Plots

Green = Standard Curve

Blue = Samples

Red = No template control

 

Standard Curve

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qPCR – Ava’s RLO Transmission Samples Re-runs

The final plate from my runs of Ava’s samples had a bad standard curve, so I’m re-running them. Additionally, Ava has asked me to add some additional samples. Here are the additional samples:

51
120
135
15:11-86
15:11-30

Sample 120 did not have any volume left (because I used the rest of that sample during the previous qPCR), but, for kicks, I added 5uL of nuclease-free water to it and ran it anyway.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170406_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on the Lisa’s development of the withering syndrome qPCR assay.

Results:

qPCR Report (PDF): Sam_2017-04-06 07-53-11_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-04-06 07-53-11_CC009827.pcrd

Curve looked good. Will let Ava know that all the samples are finished.

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qPCR – Capstone RLO Viability DNased RNA

Need to verify that the DNased RNA I made previously does not have any detectable gDNA present.

Ran the withering syndrome qPCR assay on the DNased RNA.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate. As such, the number of samples required to qPCR runs.

Master mix calcs are here (Google Sheet): 20170406_qPCR_WSN1_capstone

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

Baseline threshold was manually set to 580, as previously determined by Lisa.

Results:

qPCR Report (PDF): Sam_2017-04-06 10-01-23_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-04-06 10-01-23_CC009827.pcrd

qPCR Report (PDF): Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pdf
qPCR Data File (CFX96): Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd

Well, some samples came up positive for residual DNA. The samples that came up positive are all three dilutions of the RLO used for initial infection of the abalone.

This makes things interesting to deal with. Seeing that no other samples have detectable DNA suggests that those samples are fine to move forward with for reverse transcription. However, it’s unlikely that the DNase treatment only worked on a subset of a samples, since it was distributed via a master mix.

Regardless, there isn’t any additional RNA to work with. So, I’ll put the samples that came up positive through a second round of DNase treatment. Addtionally, I may dilute them slightly to avoid complications from accumulation of too much DNase buffer, due to leftover buffer from the first round…


Amplification Plots from Sam_2017-04-06 10-01-23_CC009827.pcrd

Green = p18RK7 standards
Blue = samples
Red = No template control

 

Standard Curve from Sam_2017-04-06 10-01-23_CC009827.pcrd

 

 

Amplification Plots from Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd

 

 

Standard Curve from Sam_2017-04-06 11-36-53_CC009827_capstone_RLO_viability_WSN1.pcrd

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