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PCR008: Testing different qPCR mixes on crystal PCR (open)
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PCR008: Testing different qPCR mixes on crystal PCR (open)

Friday, 8/20/2021AuthorsBackground and GoalRun 1Recipe: Takara Primescript IIIRecipe: NEB Luna 2X mix 1 stepRecipe: NEB Luna 4X mix + UDGPCR008_Tammy's_crystalPCR_recipe.jpgQuantaBio Perfecta Mixnaica multiplex 5XTable4Table1Table7Tuesday, 8/24/2021PCR008_R1_crystal_summary.pngPCR008_R1_crystal_droplet_calling.pngThursday, 9/9/2021PCR008 Run 2Table2Table3PCR008_R2_chip_setupQuantaBio qScript® XLT One-Step RT-qPCR ToughMix®RT-qPCR program (from QuantaBio qScript® XLT One-Step RT-qPCR ToughMix®)Friday, 9/10/2021Results from PCR008_R2PCR008_R2_Blue.pngPCR008_R2_Green.pngdroplets_by_colour.pngdroplets_by_concentration.pngProbe concentration comparisons
Friday, 8/20/2021
Authors
This is a copy of the original notebook created by Trevor Ho and Nahuel Moreno in Nick Gilbert group (https://chromatinlab.org/) for the COVID Wastewater Scotland project (https://biordm.github.io/COVID-Wastewater-Scotland/)
Background and Goal
Objective:
Run 1
We intend to test droplet formation on the stilla ddPCR. Our Naica mastermix hasn't arrived and we will therefore test our NEB Luna, Luna+UDG and Takara Primecript III. We will load four blank samples with only fluorescein, and four more with probes, primers and sample. We are taking some samples of ours and our probes for loading two wells with Tammy's Naica master mix.
PCR008_Tammy's_crystalPCR_recipe.jpg
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Quanta bio
Sample dilutions:
For obtaining 50:50 E484K:E484WT use 10 μL of each sample and mix them together, then take 1 μL for loading into the PCR mix.
We ended up not doing this, but it is useful for future experiments. We used DNA samples instead as the Naica kit was not an RT-qPCR mix.
DNA sample dilutions:
Chip arrangement:
After obtaining the results, we could see that droplet formation was indeed much better using either the PerfeCTa multiplex mix or the naica 5X qPCR mix.
Tuesday, 8/24/2021
Reporting summaries:
PCR008_R1_crystal_summary.png
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PCR008_R1_crystal_droplet_calling.png
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Observations:
1.
PCR mixes were really the determining factor in droplet formation.
2.
Unsure if the PCR were successful in any of these mixes. We were expecting some droplets to be brighter and others to be darker.
Either, all droplets were saturated with both templates or PCR failed in all droplets. We should have included a negative control with just mixes to be able to set a baseline for comparison.
Thursday, 9/9/2021
PCR008 Run 2
The QuantaBio qScript® XLT One-Step RT-qPCR ToughMix® has finally arrived, we will continue the experiments with Stilla ddPCR.
We are interested in measuring four things:
●
dropplet formation
○
This will be measured passively when the PCR is run by observing the formation of dropplets in the wells.
●
negative vs possitive signal dropplets
○
We will run an NTC for comparison and we will hope that highly diluted samples will display both possitive and negative dropplets.
●
sensitivity of multiplexing assays
○
A mix of 50:50 WT and MUT template will be serially diluted and run. Ideally, the probes should be able to outcompete each other in the right conditions.
●
specificity of the probes.
○
Both probes will be run simultaneously on each individual template to detect unspecific binding.
​Inaccessible Entry determined that the most promissing probes are the ones for K417T, therefore we will be using those for this assay, as well as the IVT samples from ​Inaccessible Entry.
Sample dilution:
We will start by diluting the samples to 1 ng/μL, then do serial 5-fold dilutions from there.
An Opal chip will be used for the experiment.
Reactions for the probe competition assay will take 1 μL of RNA sample, while the sample mixture assay will take 1 μL of each sample, totalling 2 μL of the reaction. The NTC runs in the first column of wells in the Opal chip, with the sample mixture assay, and therefore 2 μL of water will be loaded in it.
The Welcome Trust centre closed at 4:30 so the machine was left overnight and the chip will be imaged on the following day.
Friday, 9/10/2021
Results from PCR008_R2
PCR008_R2_Blue.png
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PCR008_R2_Green.png
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droplets_by_colour.png
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droplets_by_concentration.png
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We are observing that the mutant probe binds to the WT template when the template concentration per droplet decreased. However, we did not see the same effect from WT probe on mutant template. This was very strange and was the exact opposite of the results obtained from ​Inaccessible Entry .
One observation was that as the template concentration decreased, we saw that mutant probe started to give signal with WT template. Whereas, when WT template concentration was high, none of the droplets gave strong signal from mutant probe binding.
One potential reasons we thought of was the may be the probe to template ratio could be modified / optimized.
Looking back:
Also:
• Recommended primer and probe concentrations on the naica® system are 0.25 to 1 µM.
We can conclude that the best qPCR mix is the QuantaBio qScript® XLT One-Step RT-qPCR ToughMix®.
We will proceed in future experiment with this.
This experiment is now closed.

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