Earlier today, I created dilution series of the following two linearized plasmids to develop qPCR assays:
Master mix calcs: 20151106 – qPCR RLOv Standard Curves
All samples were run in triplicate on a CFX96 thermal cycler (BioRad).
Cycling params, plate layout, etc. can be found in the qPCR Report (see Results below).
qPCR Data File (CFX96): Sam_2015-11-06 18-17-41_CC009827.pcrd
qPCR Report (PDF): Sam_2015-11-06 18-17-41_CC009827.pdf
DNA Helicase Curve
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 standard curve dilutions to use for qPCRs with the following two linearized plasmids:
Used a spreadsheet developed by Nate many moons ago to determine necessary volumes based on plasmid size to obtain desired copy numbers.
Dilutions were made in TE Buffer.
DNA helicase dilutions (Google Sheet): 20151106 – Dilution table RLOv_DNA_helicase_qPCR_Standard_Curve
Head-to-tail dilutions (Google Sheet): 20151106 – Dilution table RLOv_head_to_tail_qPCR_Standard_Curve
Ran conventional PCR to amplify the full length abalone withering syndrome 16s product for subsequent cloning. Used four different DNA plasmid preps as template, for comparison purposes. Plasmid preps used were:
- p16RK7 (from 20120718)
- p18RK7 (from 20120718)
- pWC8 (from 20120718)
- p16RK7 A (from 20131106)
Master mix calcs are here: 20131203 – cPCR WS Full Length
All reactions were run in duplicate.
40 cycles of:
- 95C – 15s
- 55C – 15s
- 72C – 2m
Ran 20uL (of 50uL reaction) of each sample on 0.8% TBE gel.
Lanes (left ro right):
1 – Hyperladder I (Bioline)
2 – p16RK7 (from 20120718)
3 – p16RK7 (from 20120718)
4 – p18RK7 (from 20120718)
5 – p18RK7 (from 20120718)
6 – pWC8 (from 20120718)
7 – pWC8 (from 20120718)
8 – p16RK7 A (from 20131106)
9 – p16RK7 A (from 20131106)
10 – NTC
11 – NTC
Expected a band of ~1500bp. Bands of all samples (except pWC8) run at ~1500bp. pWC8 runs a bit higher and will not be used for cloning, as part of our troubleshooting the failure of our plasmid qPCR standard curve for withering syndrome.
Inoculated 5mL of LB + Amp (100ug/mL) in 50mL conicals with one of the following Withering Syndrome clones frozen stocks (located in -80C box called “WS RLP Plasmids”):
- PWC8 WFS-RLP pCRII (from 4/13/01)
- p16RK3 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
- p16RK7 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
- p18RK7 WFS-RLP rDNA pCR2.1 (from 4/15/01)
Incubated O/N, 37C, 200RPM. Will isolate plasmid DNA tomorrow.
We identified a usable clone for the RLP qPCR assay that contains the entire GenBank sequence (AF133090), instead of the partial (and incorrect) clone that Lisa and I had used previously (see 20120323); leading to a month’s worth of qPCRs that failed to produce the expected, and desired, results. This clone is vector pCR2.1 (Invitrogen) containing the full GenBank sequence, AF133090).
Since the p16RK3-C clone failed to grow yesterday, decided to make cultures from all three frozen stocks of p16RK3 to ensure that at least one will grow.
Three 5mL O/N cultures in LB+Amp (100ug/mL) were inoculated directly from the frozen stocks and grown at 37C on a rocker in a 15mL conical tube.
All three cultures failed to grow. Left cultures in incubator on rocker. Will make some LB and LB+Amp plates and try streaking out all three frozen stocks. Also will try growing the frozen stocks in liquid LB (no Amp) O/N in hopes of getting a culture for mini prep.