Tag Archives: p16RK7

qPCR – Withering Syndrome ddPCR Comparison

Performed qPCR on the same exact samples previously sent to Alice Nguyen at the Marine Science Institute for digital droplet PCR (ddPCR) to compare our results to hers. These samples were previously quantified as part of the Ab Endo Project (Ab Endo 2011 Water Filter DNA samples). Here’s the list of samples that were sent to (and back from) Alice:

  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 p16RK7 (from 20120730).

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

Results:
qPCR Report (PDF): Sam_2015-04-15 15-38-13_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-04-15 15-38-13_CC009827.pcrd

Table – Summary of qPCRs and ddPCR data

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

The table certainly shows some inconsistency, both between ddPCR and qPCR. Additionally, there is also inconsistency between the initial qPCRs and today’s qPCRs. Due to the limited sample volumes remaining for these sub-samples that were sent to Alice and then returned to us, I will repeat the qPCR using the stock DNA and see how that compares.

Share

qPCR – Withering Syndrome qPCR Assay Validation: Reproducibility (CFX 96)

Ran qPCR for the reproducibility aspect of the WSN qPCR Assay Validation. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the Results (see below).

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Samples used for “low”, “medium” and “high” copy numbers for each sample type are below, with expected fold copy number (based off of previous qPCRs):

Feces

Low: R4E 4/17/09 – 10^0

Med: R3E 7/23/09 – 10^3

High: R4E 7/23/09 – 10^4

Tissue

Low: 09:16-18 – 10^1

Med: 09:16-22 – 10^2

High: 09:20-11 – 10^5

Water

Low: 494:11-11 – 10^0

Med: 494-11-12 – 10^2

High: TAF SD A2 – 10^3

Results:

qPCR Data File (CFX96): Sam_2012-10-22 16-16-19_CC009827.pcrd

qPCR Report (PDF): Sam_2012-10-22 16-16-19_CC009827.pdf

Everything looked good except for the Low Feces sample which didn’t produce any amplification. Will identify another sample to use for the Low Feces sample.

Share

qPCR – “Big Reds” Tank Water Filter DNA (from earlier today)

Ran qPCR on the water filter extractions done earlier today. Master mix calcs are here. Plate layout, cyclilng params, etc can be found in the Results (see below).

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Results:

qPCR Data File (CFX96):Sam_2012-10-19 11-33-26_CC009827.pcrd

qPCR Report (PDF):Sam_2012-10-19 11-33-26_CC009827.pdf

Share

qPCR – Withering Syndrome qPCR Assay Sample Checks

Ran qPCR on water filter samples isolated on 20120127 in an attempt to find water samples that have a higher copy number than Nate’s existing samples for use in the withering syndrome qPCR assay validation.

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

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Results:

qPCR Data File (CFX96):Sam_2012-10-17 15-49-02_CC009827.pcrd

qPCR Report (PDF):Sam_2012-10-17 15-49-02_CC009827.pdf

Everything looked good. Will use sample TAF SD A2 as the high copy number water sample for the withering syndrome qPCR assay.

Share

Bacterial Cultures – Withering Syndrome Clones

Inoculated 5mL of LB + Amp (100ug/mL) in 50mL conicals with one of the following Withering Syndrome clones frozen stocks (located in -80C box called “WS RLP Plasmids”):

  • PWC8 WFS-RLP pCRII (from 4/13/01)
  • p16RK3 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
  • p16RK7 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
  • p18RK7 WFS-RLP rDNA pCR2.1 (from 4/15/01)

Incubated O/N, 37C, 200RPM. Will isolate plasmid DNA tomorrow.

Share