Tag Archives: 15:30-04

qPCR – Pinto Abalone DNA with WSN1 and RLOv DNA Helicase

Ran qPCRs using both WSN1 and RLOv DNA Helicase primers on the pinto abalone DNA isolated earlier today, as well as one additional sample that Sean Bennett had previously isolated DNA from: 15:6-26E

RLOv helicase standard curve is from 20151106.

WSN1 standard curve is p18RK7 from 20170703

All samples were run in duplicate.

Master mix calcs (Google Sheet): 20171226 – qPCR Pinto WSN1 & RLOv DNA Helicase.

Plate layout, cycling params, etc. can be seen in the qPCR Reports (see Results below).

Results:

RLOv helicase qPCR Report (PDF): Sam_2017-12-26 13-19-51_CC009827_RLOv_helicase.pdf
RLOv helicase qPCR File (CFX): Sam_2017-12-26 13-19-51_CC009827_RLOv_helicase.pcrd

WSN1 qPCR Report (PDF): Sam_2017-12-26 13-19-51_CC009827_WSN1.pdf
WSN1 qPCR File (CFX): Sam_2017-12-26 13-19-51_CC009827_WSN1.pcrd

Both standard curves are acceptable (see images below).

No amplification with either primer/probe set in the following samples:

  • 15:30-01
  • 15:30-04

I believe these are both “Control” samples (i.e. unexposed) and no amplification was expected.

All other samples amplify. See qPCR Reports for copy numbers.

RLOv Helicase Amplification & Standard Curves

WSN1 Amplification & Standard Curves

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DNA Isolation & Quantification – Pinto Abalone

Isolated DNA from the following pinto abalone (Haliotis kamtschatkana) digestive gland tissues (stored in ethanol), collected by Sean Bennett as part of his Capstone project:

Accession Weight(mg)
15:30-01   194
15:30-04   67
15:31-01   34
15:31-02   107
15:31-03   83
15:31-04   80

Tissue was weighed and then DNA extracted.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Used the Roberts Lab Qubit 3.0 and the Qubit hsDNA Kit (high sensitivity). Used 1uL of template for all samples.

Samples were stored at -20C in FSH240 in the “Pinto Transcriptome DNA” box.

Results:

All samples have DNA.

Concentrations (Google Sheet): 20171226_qubit_DNA_pinto_ab

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