Tag Archives: p18RK7

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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Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution

We are virtually out of the withering syndrome qPCR standard curve plasmid dilutions and I need to send some to Colleen Burge at Univ. of Maryland Baltimore County. As such, I’ll need to make a fresh dilution series and test it out prior to sending to her.

Used NcoI-linearized p18RK7 abalone withering syndrome plasmid (from 20120726) to make a fresh dilution series.

Since it’s been nearly 4yrs since the plasmid was prepared, I decided to re-quantify the DNA using a Qubit3 (Life Technologies) in the Roberts Lab. Used the dsDNA BR Qubit reagents.

SAMPLE CONCENTRATION (ng/μL)
p18RK7 NcoI-linearized 29.0

The result is pretty close to what had been measured 4yrs ago (~26.1ng/μL – difference of  ~10%), so that’s good to see.

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 – 2011 Ab Endo Water Filters

Master mix calcs are here: 20150622 – qPCR WS Ab Endo H2O Filters

All samples were run in duplicate. See the qPCR Report (in Results below) for plate layout, cycling params, etc.

Standard curve was p18RK7 from 20120731.

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2015-06-22 14-25-45_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-06-22 14-25-45_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet.

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Samples for Abalone Withering Syndrome ddPCR

I selected the following samples (Ab Endo 2011 Water Filter DNA samples) to send to Alice Nguyen at the Marine Science Institute for digital droplet PCR (ddPCR):

  1. CI SRI CP 1A (0 copies)
  2. CI SRI CP 2B (highest)
  3. CI SRI CP 2A (0 copies)
  4. CI SRI CP 1B (high)
  5. CARMEL +500M 1 (medium)
  6. CARMEL +500M 2 (low)

10μL of each sample was sent. Tubes were labelled with “DNA #”. The ‘#’ corresponds to the number in the list above.

10μL of each of p18RK7 qPCR standards (from 20120730) were sent.

Two sets of WSN1 F/R working stocks (10μM) were also sent.

Also sent the QX200 Droplet Generation Oil for EvaGreen Kit (BioRad) that we ordered.

Shipment was on “wet” ice.

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qPCR – Withering Syndrome ddPCR Comparison Repeat

Repeated the qPCR from yesterday due to inconsistent results when compared to the previous qPCR data (see table in yesterday’s notebook entry), but used the stock DNA for template.

These samples were initially quantified as part of the Ab Endo Project (Ab Endo 2011 Water Filter DNA samples). Here’s the list of samples:

  1. CI SRI CP 1A
  2. CARMEL +500M 2
  3. CI SRI CP 2B
  4. CI SRI CP 2A
  5. CI SRI CP 1B
  6. CARMEL +500M 1

Master mix calcs are here: 20150415 – qPCR WS Ab Endo vs ddPCR

Standard curve was p18RK7 (from 20120730).

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

Results:
qPCR Report (PDF): Sam_2015-04-16 12-14-37_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-04-16 12-14-37_CC009827.pcrd

TABLE – Summary of qPCRs and ddPCR data

Sample Number Sample Name Initial qPCR Copies ddPCR Copies Yesterday’s qPCR Copies Today’s qPCR Copies
AB01 CI SRI CP 1A 0 3.8 0 0
AB02 CARMEL +500M 2 1.57 8.7 1.3 1.05
AB03 CI SRI CP 2B 147 104 59.2 201
AB04 CI SRI CP 2A 0 175 83.1 0.68
AB05 CI SRI CP 1B 74.8 2.8 0 101
AB06 CARMEL +500M 1 1.08 4.9 0 0.37

Today’s qPCR data matches up very well with the initial qPCR data.

Clearly, there is something odd about the aliquots that were sent to Alice (and back) for use in ddPCR. I can’t really fathom what happened. Will contact Alice and see if she’d like to try out new aliquots of these samples to see how the ddPCR turns out (again).

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