Since we have a working qPCR for detecting the withering syndrome bacteriophage (RLOv), Carolyn wanted to see how detection/quantification compared to withering syndrome detection/quantification on water samples collected from various farms and their nearest wild abalone site.
DNA samples used were extractions from water filters collected for the Ab Endo Project in 2011.
Overall, things are looking better than yesterday’s run: better reps, better efficiency and better R^2. Will move forward with beginning to validate this qPCR assay, as well as use for some other sample analysis that Carolyn has in mind (comparing RLOv vs. WS levels in abalone collected from wild sites in California).
The efficiency & R^2 values look pretty solid, but the spacing between the 300 copy (Cq ~ 32 in the amplification plot) and the 30 copy (Cq ~ 34 in the amplification plot) samples is a bit too tight for my liking. Additionally, the reps for the 3 copy sample are very poor.
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).
As part of the qPCR validation for the withering syndrome phage (RLOv) project, I needed to identify (and, eventually locate) samples that are infected with varying levels of RLOv. This is probably the most time consuming aspect of the project.
The Roche protocol recommends using only 10pg of plasmid DNA for probe labelling. As such, all three probes were diluted 1:10,000. A 1:1000 (999μL H2O + 1μL of plasmid) was made first. Then a 1:10 dilution was made (90μL H2O + 10μL from 1:1000 dilution of plasmid).
Additionally, I ran half reactions to conserve kit components. Roche recommends 50μL reactions; I ran 25μL and scaled all components appropriately.
All reactions were set up on ice and run in 0.2mL strip-cap PCR tubes.
After the PCR, 5μL of each reaction was run on a gel.
Hyperladder I (Bioline)
PCR DIG probe labelling products run on 1.1% agarose 1x TBE gel stained w/EtBr. A ‘+’ indicates DIG reaction, while a ‘-‘ indicates no DIG in reaction.
Two reactions were run for each plasmid: one with the DIG label (indicated by a ‘+’) and one without (indicated by a ‘-‘). If the labeling was successful, the PCR products from those reactions containing DIG will be larger (i.e. migrate slower) than those without. That is exactly what we see in each of the three potential ISH targets.
So, we now have three ISH probes ready for action! Will proceed with making fresh ISH buffers and ISH.
Probes were transferred to 0.5mL snap cap tubes and stored in my -20C box.
Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).
Vol for 1.5μg (μL)
H2O to 40μL
Digestion Master Mix
SINGLE REACTION (μL)
x 5.5 (μL)
10x Buffer 3.1 (NEB)
Add 10μL to each tube
Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.
Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.
Hyperladder I (Bioline)
U = Undigested; Bam = BamHI digest
Besides the funky way this gel ran, the digests look to be complete.
Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).
Each clone’s sequence matches that of the source sequence, so we’re good to go!
Will proceed with dye-based quantification of each plasmid. Will then proceed with developing ISH probes (membrane genes 1 & 2, tail fiber gene) or qPCR standard curves (DNA helicase, head-to-tail).
In the alignments below, the reference sequence is highlighted in light yellow. The two electropherograms are align below the reference. The grey line in the consensus sequence indicates any sequence disagreements by placement of a black mark at the position. However, the sequences all match, so there are no black marks in the regions between the identified vector sequences (red annotations below each electropherogram).
Submitted ~500ng of each plasmid in a final volume of 15μL (including primer). Each clone will be sequenced from each direction with M13F (-21) (25pmol; 2.5μL of 10μM stock) and M13R primers (25pmol; 2.5μL of 10μM stock) for a total of 10 sequencing reactions: