Carolyn & Stan Langevin have agreed that the DNA helicase qPCR should be tested on 10 black abalone DNA extractions that fall into multiple levels of the Friedman Lab’s withering syndrome histology scoring.
50% of the RLO/RLOv 0 score samples are positive for RLOv DNA helicase. Will talk to Carolyn to see if she has withering syndrome qPCR data for these samples to compare RLOv-positive samples with WSN-positive samples. If not, will run withering syndrome qPCR.
All RLO/RLOv 1 & 2 scored samples are positive for RLOv DNA helicase
All RLO/RLOv 2 scored samples come up before the standar curve; these should be diluted and re-run.
Standard curve isn’t perfect (the 3 copy sample is throwing it off).
Samples were run on a 0.8% agarose 1x TBE gel, stained with ethidium bromide.
Amplification looks great. No amplification in no template controls (NTCs). Excised bands and purified products using Ultrafree DA Spin Columns (Millipore). Samples will be stored @ 4C until I am able to clone them for sequencing.
Gel image showing excised bands. And, it’s a complete hack job, which is embarrassing…
ORANGE – WSN1; BLUE – DNA Helicase; GREEN – Head-to-tail
The results look great! The two RLOv (phage) primer sets only amplify in the sample that has histological confirmation of the presence of phage (06:6-54). They do not amplify in the RLO-only (no phage; 06:5-6) sample, demonstrating that these two primer sets are indeed specific to the phage and don’t amplify the RLO.
The withering syndrome primers (WSN1) were run to confirm that there aredetectable levels of RLO in both the RLOv & RLO samples, to further support the evidence showing the specificity of the two phage primer sets.
Will use the two RLOv primer sets in a conventional PCR for cloning/sequencing and development and validation of a qPCR standard curve.
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).