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.

DNA Quanting – Black Abalone Digestive Gland DNA

Quantified gDNA from the following samples in preparation for high-throughput sequencing by Stan Langevin’s group. Quantification was done using Pico Green, Tecan plate reader and Magellan 6 software. The r^2 value of the standard curve was 0.9986 and replicates showed little variation.

Concentrations below are the mean of three replicates and are in ng/uL.

  • 06:5-28 – 42.4
  • 06:6-41 – 60.3
  • 06:6-44 – 57.1
  • 06:6-53 – 72.1
  • 06:6-54 – 40.1
  • 06:6-55 – 40.5
  • 06:6-66 – 40.6

Stan Langevin has requested at least 50ng of DNA from each sample. I will aliquot ~100ng of each sample into individual tubes. He will be picking up aliquots tomorrow morning for sequencing on the MiSeq (Illumina).