Stained DNA Research Blog

  • bkamermans8

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

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).
  • bkamermans8

Updated: Feb 19

Scanning electron microscopy is being used by the SSRC to determine species of dinoflagellates in the Bellingham Bay and Nooksack River.

Images below were taken at Western Washington University (WWU) with the assistance of Dr. Michael Kraft. Due to COVID-19 restrictions, all sessions are virtual. Thankfully, we can run the instrument from the SSRC. Although, I do miss touching the instrument.

These images were taken with a Tescan VEGA3 microscope at 10.0 kV.

The above SEM image is of Alexadrium fundyense cells that have been fixed using 2% glutaraldehyde and 1% osmium tetroxide (OsO4), and freeze dried for 5 minutes on glass slides.

The above SEM image is of a Prorocentrum cell cultured in L1 broth and collected from the Bellingham Bay Buoy (Se'lhaem) in October 2020. The sample has been fixed using Lugol's, 2% glutaraldehyde and 100% hexamethyldisilazane (HMDS), and airdried for 5 minutes on glass slides.

The use of OsO4 is very toxic and hard to handle. Ideally, we would use a Critical Point Drying (CPD) apparatus or machine to achieve the temperature/pressure combination to completely dehydrate a small specimen for SEM work. However, due to COVID-19 restrictions, we are unable to access this kind of equipment at WWU. HMDS (hexamethyldisilazane) may be used to replace the CPD step in sample preparation of cells with "armor" (for instance the Alexandrium are “armored dinoflagellates” meaning it has thecal plates made of cellulose surrounding the cell like armor) for SEM imaging. All HMDS steps need to be carried out in the fume hood wearing the necessary personal protection gear as it is also highly toxic.

I hope to acquire more quality images with better preservation in the future. For instance, I would have liked to have seen flagella in the image of the Prorocentrum cell. In future experiments, I would like to try filtering the environmental samples, instead of using a centrifuge. I think Lugol's, 2% glutaraldehyde and 100% hexamethyldisilazane (HMDS) steps can easily be done with a filter tower in the fume hood. I cannot imagine a safe way to handle the OsO4 in the fume hood and with a filter tower, at this time.