Tag Archives: 2_ORF25_121_CAB

qPCR – Withering Syndrome Phage

Ran qPCR using the following primer sets designed off of the potential WS phage sequence that Stan Langevin has sequenced:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Templates tested were abalone digestive gland gDNA:

  • 06:6-41 (4/7/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 06:6-53 (4/9/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 08:4-1 – Positive for withering syndrome only – no phage.
  • UW08:22-11A – Pinto abalone naive for both withering syndrome and phage.

Master mix calcs are here: 201400820 – qPCR WS phage

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

Results:
qPCR Report (PDF): Sam_2014-08-20 14-29-56_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-08-20 14-29-56_CC009827.pcrd

Great news! No amplification in the 08:4-1 (positive for WS, but naive for phage)!! These results strongly suggest that these primers are specific for the WS bacteriophage! This is really cool and exciting. Next steps will be to confirm via in-situ hybridization (ISH).

Additional summary of the results:

Primer set ORF25_CSF shows the highest sensitivity.

Primer set ORF20_CSF fails to amplify anything in 06:6-53

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PCR – Withering Syndrome Phage

We received MiSeq data back from Stan Langevin (samples submitted 20140717) and he believes he has sequenced the entire WS phage. Carolyn and Colleen designed some primers on two of the open reading frames annotated by Stan. Ran PCR with the three primer sets to test out:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Master mix calcs are here: 201400813 – PCR WS phage

Cycling params:

Ran samples on 1.2% 1x TBE + EtBr.

Results:

Ladder: O’GeneRuler 100bp DNA Ladder (ThermoFisher)

Good amplification from all three primer sets. The pinto abalone sample (UW08:22-65) that should be naive for withering syndrome and phage did not amplify as expected.

Excised bands from each primer set in the 06:6-41 group and purified using Ultrafree DA spin columns (Millipore). Will save for potential cloning usage, depending on future results.

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