Tag Archives: p18RK7

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 – 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 – 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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qPCR – DNased RNA from Abalone Water Filters (from earlier today)

Prior to creating cDNA, need to verify that the DNased RNA from earlier today doesn’t contain any detectable RLO DNA.

Master mix calcs (Google Sheet): 20161128 – qPCR Water Filter DNased RNA

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_2016-12-07 09-10-07_CC009827.pdf
qPCR File (CFX96):Sam_2016-12-07 09-10-07_CC009827.pcrd

Standard curve looks good.

No samples amplified. This suggests that there is no detectable DNA in any of the DNased RNA samples. Will proceed with making cDNA.

 

 

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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 (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_2016-11-28 11-02-46_CC009827.pdf
qPCR Data File (CFX): Sam_2016-11-28 11-02-46_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve to past versions of the curve. Will put new curve in the standard WS plasmid curve box in FSH240 fridge.

 

ALL MASTER MIXES

 

 

 

MASTER MIX #1


 

MASTER MIX #2

 

 


MASTER MIX #3

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Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

Lisa recently ran a qPCR and noticed that the standard curve had drifted quite a bit and was no longer usable, so I’m making more.

Used NcoI-linearized p18RK7 abalone withering syndrome plasmid (from 20120726) to make a fresh dilution series. The plasmid was most recently re-assessed and successfully used for a new standard curve on 20160316. As such, I re-used the dilution calculations from 20160316 (see sheet below).

I prepared the dilution series using IDTE (pH=8.0; IDT) Buffer. The dilution worksheet below was set up by Nate Wight many, many, moons ago.

Dilution calcs (Google Sheet): 20160316 – WS p18RK7 plasmid dilution table

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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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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 (Google Sheet): 20150316 – 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_2016-03-16 17-04-05_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-16 17-04-05_CC009827.pcrd

Overall, the curve looks good and has very comparable Cq values at each dilution of the curve. Will use this for future withering syndrome qPCR assays and will send an aliquote of each dilution to Colleen Burge.

MASTER MIX #1


 

MASTER MIX #2

 


MASTER MIX #3

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