Tag Archives: WSN1

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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qPCR – Ava’s RLO Transmission Samples

Ava provided me with a list of samples that needed to be qPCR’d (Google Sheet): qPCR redos 30117.xlsx

Here’s a list of samples that had no liquid left in them (likely due to evaporation). I added 5uL of nuclease-free water to each sample in hopes of gleaning some data from them:

14
22
37
38
46
48
49
50
52
55
61
65
116
127
149
152
155
157
158

The following samples are samples that I used the remainder of them for these qPCR reactions:

60F1
120
136

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170322 – qPCR WSN1 Ava Samples 01

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:

All but the final plate look good (standard curve-wise). Will re-run last plate next week.

qPCR Report (PDF): Sam_2017-03-22 07-24-02_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 07-24-02_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 08-54-50_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 08-54-50_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 10-25-58_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 10-25-58_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 11-54-57_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 11-54-57_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 13-23-37_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 13-23-37_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 14-51-55_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 14-51-55_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-22 16-19-59_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-22 16-19-59_CC009827.pcrd

qPCR Report (PDF): Sam_2017-03-23 06-54-02_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-23 06-54-02_CC009827.pcrd

 

NOTE: This needs to be re-run, due to a wonky rep of one of the points of the standard curve.
qPCR Report (PDF): Sam_2017-03-23 08-24-59_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-03-23 08-24-59_CC009827.pcrd

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qPCR – LCM DNA

Ran three primer sets on laser capture microscopy (LCM) DNA samples from 2005 and 2007. Ran the following primer sets:

  • WSN1 (detects RLO)
  • RLOv_helicase (detects RLO phage)
  • XenoCal_prophage

The DNA samples were provided to me by Lisa. I’m not entirely sure of their history:

 

Master mix calcs (Google Sheets):

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

Standard curves:

Baseline threshold was manually set to 580 for the WSN1 samples, as previously determined by Lisa for this assay.

Baseline threshold was manually set to 580.5 for the RLOv DNA helicase samples, as previously determined by me on 20160128.

 

Results:

WSN1:

 

RLOv DNA helicase:

 

XenoCal prophage:

 

Summary table of all three genes in each sample. Unfortunately, I don’t fully understand the sample name nomenclature, so I can’t really come to any conclusions about the data. Will pass along to Carolyn, Lisa, and Stan.

It’s also important to note that, due to low sample volume, I did not quantify these samples. This is important because any samples listed below that are negative for all three genes can not be conclusively declared “negative”, since we can’t rule out the possibility that they simply lack any DNA.

Presumably they were quantified after their initial extraction?

SAMPLE WSN1 RLOv DNA HELICASE XC PROPHAGE
LCM New RLO 09 + + +
LCM ST RLO 09 - - -
LCM New 08:30-5 B + + +
LCM New 08:30-5 - - -
LCM ST 08:30-3 - - -
LCM WS RLO + - +

 

STANDARD, AMPLIFICATION, & MELT CURVES

 

WSN1

 

 


 

 

RLOv_DNA_helicase

 


 

 

XenoCal prophage

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qPCR – Water Filter cDNA for RLO Viability Assessment

Ran qPCRs on the cDNA I made earlier today to determine if there’s any detectable RNA in any of these water filter samples.

Master mix calcs (Google Sheet): 20161208- qPCR WSN1

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

Standard curve was the p18RK7 curve made on 20161128.

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

Results:
qPCR Report (PDF): Sam_2016-12-08 09-14-38_CC009827_cDNA_WSN1.pdf
qPCR File (CFX96): Sam_2016-12-08 09-14-38_CC009827_cDNA_WSN1.pcrd

Original qPCR File (CFX96): Sam_2016-12-08 09-14-38_CC009827.pcrd

Standard curve looks good.

The following cDNA samples had detectable amplification:

  • T1A
  • T1B
  • T3A
  • T3B

I believe that the labelling scheme represents T1 = Day 1 in water, T3 = Day 3 in water.

These results suggest that the RLO is viable outside of the abalone host for at least three days, but not >= 7 days, although the values are below the theoretical qPCR limit of detection. These results will likely be used to help Lisa with experimental design for a more involved assessment of RLO viability in the water column.

I’ve added the data to Lisa’s spreadsheet (Google Sheet: RLO viability) in the “Expt 1″ worksheet.

Update after talking to Lisa: The water was shipped from a California abalone farm O/N, so T0 = 24hr water. The Control water samples were sea water from our basement facility, not from California.

The fact that there is no amplification at T0 is a bit surprising and possibly suggests that RLO viability outside of the host is on the magnitude of hours, not days…

 

 

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