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
Ran samples on 1.2% 1x TBE + EtBr.
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.