Tag Archives: GoTaq qPCR Probe Master Mix

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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qPCR – CDFW White Abalone Samples (RLOv DNA helicase)

The samples that CDFW sent us earlier were previously checked for RLO presence with the withering syndrome qPCR assay.

Standard curve was from 20151106.

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): 20170420 – qPCR RLOv DNA Helicase

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

Baseline threshold was manually set to 580.5, as previously determined.

Results:

qPCR Report (PDF): Sam_2017-04-20 07-50-18_CC009827.pdf
qPCR Data File (CFX): Sam_2017-04-20 07-50-18_CC009827.pcrd

Standard curve looks good and all samples provided come up positive for RLOv DNA helicase.

I’ve compiled the raw data of both the WSN qPCR and this in this Google Sheet: 20170420_CDFW_White_Ab_qPCR_summary

Here’s a summary table of the results (copy numbers are mean copies from qPCR replicates):

SAMPLE RLOV DNA HELICASE (COPIES) WSN1 (COPIES)
SF16-76_DG-1  165318.58 169.25
 SF16-76_DG-2  47839.81  20.70
 SF16-76_PE-1  1036697.17 633.75
 SF16-76_PE-2  46763.60  296.83
 SF17-17  117.29  2.16

NOTE: The WSN1 copies for SF17-17 is below the accepted detection limit of the qPCR assay (i.e. < 3 copies).

Will share my notebooks and spreadsheet with Blythe at CDFW.

Amplification Plots

Green = Standard Curve

Blue = Samples

Red = No template control

 

 

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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 – RLOv DNA Helicase 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 from 20161106.

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

Baseline was set 580.5 as previously determined.

Results:
qPCR Report (PDF): Sam_2017-04-13%2016-20-54_CC009827_RLOv_helicase.pdf
qPCR Data File (CFX): Sam_2017-04-13%2016-20-54_CC009827_RLOv_helicase.pcrd

Standard curve looked good, although efficiency is pushing it on the high end.

The following samples did <em>not</em> amplify:

  • 07:12-02
  • All 08 samples.

The remaining samples all came up positive, with the 06 set being extremely hot (came up around cycle 13).

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 – RLOv DNA helicase and XenoCal prophage on Ab Endo Water Filters

Stan Langevin was interested in seeing if the RLOv (phage) and/or the prophage portal genes were detectable in water samples from Lisa’s Ab Endo project.

Ran qPCR on the following samples that Lisa selected:

DNA from water filters collected in 2010. DNA isolated 20120111:

  • CP 0M A
  • CP 0M B
  • MA 0M A
  • MA 0M B
  • PSN 0M A
  • PSN 0M B
  • RM A
  • RM B

DNA from water filters collected in 2011. DNA isolated 20140822:

  • AM Drain 2B
  • PCI SRI PC 1B

RLOv_DNA_helicase master mix calcs are here (Google Sheet): 20161213 – qPCR RLOv DNA Helicase

XenoCal prophage master mix calcs are here (Google Sheet): 20161213 – qPCR XenoCal phage portal

RLOv_DNA_helicase standard curve from 20151224.

All samples were run in duplicate. Plate layout, cycling params, etc. can be seen in the qPCR Report below.

Results:

RLOv_DNA_helicase
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pcrd

 

XenoCal prophage
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pcrd

 

  • RLOv DNA helicase amplified in all samples EXCEPT the two samples from 2011. These two samples were negative for the RLO (see Ab Endo sheet “water 2011″).
  •  XC prophage amplfied inconsistently (i.e. replicates did not match/amplify) in only three samples. Additionally, the melt curve of one of those samples differs from the other two. Based on the inconsistencies in technical reps, I should probably repeat this, but technical reps across all of the RLOv DNA helicase samples are very tight, suggesting that my technique was fine (it would be odd if my technique faltered only on ALL of the XC prophage samples)…

 

RLOv DNA HELICASE

 


 

XENOCAL PROPHAGE

 

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