Category Archives: Abalone Withering Syndrome qPCR Assay Validation

Abalone Withering Syndrome qPCR Assay Validation

qPCR – Repeat of Earlier qPCR with New Sample

Re-ran the qPCR (see earlier entry from today), due to the Low Feces samples failing to amplify. Replaced it with:

R3E 5/14/09 – 10^0

Everything else (including master mix, cycling params, etc) is all the same. See the earlier entry for details.

Results:

qPCR Data File (CFX96): Sam_2012-10-22 13-37-42_CC009827.pcrd

qPCR Report (PDF): Sam_2012-10-22 13-37-42_CC009827.pdf

Things looked good. The replacement Low Feces sample amplified.

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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.

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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

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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.

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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.

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Bacteria Cultures – p16RK3-A, B, & C Clones from 5/2004

We identified a usable clone for the RLP qPCR assay that contains the entire GenBank sequence (AF133090), instead of the partial (and incorrect) clone that Lisa and I had used previously (see 20120323); leading to a month’s worth of qPCRs that failed to produce the expected, and desired, results. This clone is vector pCR2.1 (Invitrogen) containing the full GenBank sequence, AF133090).

Since the p16RK3-C clone failed to grow yesterday, decided to make cultures from all three frozen stocks of p16RK3 to ensure that at least one will grow.

Three 5mL O/N cultures in LB+Amp (100ug/mL) were inoculated directly from the frozen stocks and grown at 37C on a rocker in a 15mL conical tube.

Results:

All three cultures failed to grow. Left cultures in incubator on rocker. Will make some LB and LB+Amp plates and try streaking out all three frozen stocks. Also will try growing the frozen stocks in liquid LB (no Amp) O/N in hopes of getting a culture for mini prep.

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