Filtering Fairhaven Dock samples - in search of dinoflagellates

https://youtu.be/8c4pd6ZbP3c

The video is of me running a field blank and sample through a filtering apparatus. The sample was collected from Fairhaven Dock.

Over the course of summer/fall 2021, we are collecting 3L of water once a week from the dock to monitor toxic algae. We are quantifying the SxtA gene produced by a genus of dinoflagellates, Alexandrium.

In the video, I run 1.5L of ultrapure water and a 'real sample' through the beta-version filtering apparatus developed by Phytoxigene. The ultrapure water serves as a field blank. Then, I run 3L of the water from Fairhaven Dock through the filer ('real sample').

Again, the filtering apparatus is a test model apparatus built by Phytoxigene.

Phytoxigene is a company that provides assays and standards for the molecular detection and quantification of biotoxin producing genes. Click here to see more from Greg Ford of Phytoxigene

We are using qPCR assays and standards developed by Phytoxigene to detect SxtA. SxtA is the initial gene in the biosynthesis of saxitoxin.

DNA copy number calculator

A good resource to calculate copy number for a strand of DNA is found here:

https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/mole[…]hermo-scientific-web-tools/dna-copy-number-calculator.html

Based on knowledge of sequence length (nucleotides; nt) this tool can help you dilute your DNA to a certain copy number (molecules) per uL.

This tool asks for the molar mass per base pair of your DNA fragment. Duewer et al. 2018 has a great explanation for the variation in mean values for nucleotides.

I want to know the exact concentration of copies/uL I have in my standard solutions to develop a dilution curve for qPCR. I want to know the lowest concentration of copies/uL I have an accurate reading.

1. work under controlled conditions

2. small vials no bigger than 100uL

3. 1uL lowest pipettor without having error in the process of pipetting

4. Do dilutions in multiples triplicates up to x18.

Serial dilutions of stock gblock DNA of Spirinchus thaleichthys

Today I am going to use the Agilent 2100 Bioanalyzer to assess the size and quality of DNA of the gblock DNA I ordered for the Spirinchus thaleichthys.

Yesterday I took the stock concentration of the gblock from IDT that was 1.84 x10^8 molecules/uL and diluted it 10x to 1 molecule/uL. Now, I want to assess the quality of the DNA through each of the dilution steps.

The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA and RNA. ... The fluorescent dye molecules intercalate into DNA or RNA strands. They are then detected by their fluorescence and translated into gel-like images (bands) and electropherograms (peaks).