Amplifcation plots and the standard curve best fit line looks really good. Efficiency is very close to 100% and the R^2 = 0.99. Additionally, virtually all of the replicates are very tight. This looks like it will be totally usable as a standard curve for developing a qPCR assay that targets the RLOv DNA helicase gene.
This curve is way wonky. Interestingly, the end-point fluroescence levels for this curve 5-fold lower than the DNA helicase curve. I’ll likely repeat this qPCR to see if these crappy results are repeatable. However, having a single qPCR assay (the DNA helicase standard curve) for RLOv detection/quantification might be sufficient, rendering a second qPCR assay unneeded.
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).
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:
A sterile pipette tip was used to inoculate 5mL of 1x LBAmp100 in a 15mL conical tube. The tubes were incubated O/N @ 37C on a rocker.
3mL of liquid culture was used as input for plasmid isolation with the QIAprep Spin Mini Kit (Qiagen) according to the manufacturer’s protocol.
1mL of each culture was combined with 1mL of 50% sterile glycerol (25% glycerol final concentration) and stored @ -80C with existing bacterial stocks.
Plasmid DNA was eluted with 50μL of Buffer EB and quantified on the Roberts Lab NanoDrop1000 (ThermoFisher) in order to have a rough idea of concentrations to submit for Sanger sequencing. A dye-based quantification will be performed after sequencing results are back in order to obtain a more accurate assessment for use in ISH and/or qPCR standard curve creation.
Quality (260/280 & 260/230 ratios) look great and yields are more than sufficient. Will prep samples for Sanger sequencing.