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
Selected colonies 1, 2 & 3 from the colony screens from 20131205 for plasmid isolation. 5mL of liquid cultures were grown O/N at 37C. 1mL of each culture was mixed with 1mL of sterile 50% glycerol solution and stored @ -80C. 3mL of each culture was used for plasmid isolation. Plasmids were isolated using the Qiaprep Mini Spin Kit (Qiagen) according to protocol. Plasmids were eluted with 50uL of Buffer EB.
I needed additional dilutions for a low curve to finish validating the Promega 2x GoTaq Probe Master Mix limit of detection. Will test the low curve before continuing the full limit of detection process.
Master mix calcs are here: 20140507 – qPCR p18RK7 low curve check
qPCR Report (PDF): Sam_2014-05-07 14-47-55_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-07 14-47-55_CC009827.pcrd
New low curve looks good. Will use this for subsequent limit of detection tests.
Ran qPCR on two new WS standard curves using the dilutions set up earlier today. Also ran undiluted digests of both plasmids as potential positive controls.
Master mix calcs are here (20140106 – qPCR RLO New Curves). See the Results below for plate layout, cycling params, etc. All samples were run in duplicate.
qPCR Report (PDF): Sam_2014-01-06 15-49-04_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-01-06 15-49-04_CC009827.pcrd
No amplification of any kind in any of the two standard curves! However, there was amplification of both of the undiluted digest samples!
Set up curve dilutions using two of the three NcoI linearized pCR2.1/WS plasmids (from 20140104). NcoI linearized pCR2.1/WS2 was not used due to insufficient quantity of DNA remaining.
Curve calculations are here (20140106 – WS QPCR tables for dilution sets)
The spreadsheet used was set up by Nate for determining quantities of DNA to use to achieve desired copy numbers at each dilution point of the curve.
The curves were diluted in new Low TE Buffer (made 10/9/2013 SJW).
Used Pico Green (Invitrogen) protocol for quantification on the Tecan plate reader. Used two standard curves for more accurate quantification; a “low” curve for the linearized plasmids and a “normal” curve for the complete plasmids. All samples and standards were done in replicates of two.
Low curve (ng): 0, 1, 5, 10, 20
Normal curve (ng): 0, 20, 40, 80
Low curve R2 = 0.99935
Normal curve R2 = 0.99738
- pCR2.1/WS1 – 32.613
- pCR2.1/WS2 – 17.131
- pCR2.1/WS3 – 72.836
NcoI Linearized Plasmids (ng/uL)
- WS1 – 3.3956
- WS2 – 1.5668
- WS3 – 18.049
Will make dilutions of the linearized plasmids to prepare qPCR standard curves.
Linearized three plasmids for use in WS qPCR standard curve NcoI. Each reaction contained the following:
10x Buffer #3 (NEB): 5uL
NcoI (NEB): 1uL
All reactions were incubated @ 37C for 1hr and then heat inactivated @ 65C for 20mins. 25uL of each reaction was run on a gel, along with 5uL of undigested plasmid to confirm digestion.
- Lane 1 – Hyperladder II (Bioline)
- Lane 2 – pCR2.1/WS-1
- Lane 3 – pCR2.1/WS-1 NcoI
- Lane 4 – pCR2.1/WS-2
- Lane 5 – pCR2.1/WS-2 NcoI
- Lane 6 – pCR2.1/WS-3
- Lane 7 – pCR2.1/WS-3 NcoI
- Lane 8 – No template control
Ran the wrong ladder. However, we see complete digestion of pCR2.1/WS-1 and WS-2. pCR2.1/WS-3 may not be complete, as evidenced by the highest molecular weight band that is present in both the undigested and digested lanes. Although this has no effect on anything, pCR2.1/WS-3 has the insert in the opposite orientation as WS-1 and WS-2, which explains the different digestion pattern. Will create standard curves from all three digestions.
Selected 10 white colonies for PCR and restreaking. Master mix calcs are here.
Ladder = Hyperladder I (Bioline)
All colonies produced a band of the expected size (~1500bp). Will select three colonies to grow up for plasmid isolation.
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.