Purpose

The purpose of these lab activities is to extract DNA from tissues and feces from Squeaky Wiggles S. Honeycomb. The quantity of genomic and metagenomic DNA will be evaluated by spectrophotometry, fluorometry, and gel electrophoresis before proceeding to the preparation of libraries for sequencing.

Skills

This lab session will help you practice the following skills:

• Reading and carefully following a DNA extraction protocol.
• Critical thinking and experimental interpretation.
• Documenting the procedure followed and results obtained.
• Finding, citing, and summarizing research from a peer-reviewed scientific journal

Knowledge

This lab session will help you gain the following knowledge:

• Familiarity with the general steps for genomic and metagenomic DNA extraction.
• Understanding of the functional roles of the reagents and conditions used for DNA extraction.
• Understanding of the advantages and disadvantages of DNA quality assessment and quantification using the NanoDrop, TapeStation, and Qubit instruments.

Tasks

Read the protocol for the QIAGEN QIAwave DNA Blood & Tissue Kit we will use to obtain total genomic and metagenomic (DNA from the microbes in an environment) DNA from samples.

We will spend time discussing the steps before beginning the extraction process.

Teams will work on individual samples, and the group will share and discuss the results.

Your goal is to use the frozen samples and total DNA to learn about the microbes and wax worm genes that could have caused the passing of Squeaky. To achieve this goal, you will work with your team to catalog, quantify, and prepare the DNA for sequencing using Oxford Nanopore Technologies (ONT). Let’s begin the investigation!

Procedure

1. QIAwave Blood and Tissue and review the DNA extraction procedure before beginning.
2. All materials will be considered biohazard and discarded in the biohazard waste bags.
3. Reagents from the kit have been prepared for you and will be available in the center bench and/or the bench you are working in.
4. Follow the protocol for each of your samples and take notes on your Google Doc if there are changes.
5. After eluting your DNA, carefully label each tube with the date, team number, and what the sample is. Then, document this on your notes along with the sample metadata, such as the type of sample and date of extraction.
   1. For example, your team may label the tube: “Worm tissue, 7/7/23, genomic DNA” or “worm feces, 7/7/23, metagenomic DNA.”
   2. Additional details and comments should be included in your notes, as they may not fit on the tube.
6. Use the NanoDrop to obtain the concentration of the DNA and absorbance ratios that can be used to assess purity.
   1. Pipette 2 μl of your elution buffer, the last buffer you used to elute your DNA, onto the pedestal of the instrument to blank the NanoDrop spectrophotometer.
   2. Pipette 2 μl of your first sample to measure the absorbance and calculate the DNA concentration. Record the concentration in ng/μl, the absorbance ratio at 260/280 nm (A_260/280), and the 260/230 ratio (A_260/230)
   3. . These values will help us assess the quality of the DNA. Pure DNA has an A_260/280 ratio of 1.8. A 260/230 ratio near 2.0 suggests nucleic acids with few contaminants from the extraction process. Review the spectrum displayed on the screen. It should be a curve with a peak at 260 nm. Note any “shoulders” you observe.
   4. Document in your notes all values in a table with columns for the sample name, concentration in ng/μl, ratios, and comments.
   5. Repeat steps 2-4 for all your samples, wiping the pedestal with a Kimwipe between the samples.
   6. Finally, measure 2 μl of your elution buffer to confirm the NanoDrop is working correctly.
7. Bring your samples to the station we will use to prepare for the TapeStation analysis. You used the NanoDrop to quantify your DNA and will now use gel electrophoresis to assess the size of the genomic DNA fragments obtained. Remember that Nanopore devices have the advantage of being able to sequence long DNA fragments; however, our DNA isolation procedure does fragment the DNA.
   1. The TapeStation requires 1 μl of DNA and 10 μl of a buffer with a fluorescent dye for each sample. A DNA ladder with DNA fragments of known sizes is used to determine the size of your DNA fragments. Thus, the first lane of the 16 in a TapeStation screen tape is used to run the ladder.
   2. Pipette carefully 1 μl of your sample in each of the tubes with 10 μl of dye. Use the P2 pipettor set to 1 μl to transfer liquids accurately, as the TapeStation is very sensitive. We will use the Genomic DNA Screen Tape and reagents.
   3. Once all our samples are loaded, the TapeStation will run for 20-30 minutes and generate an electropherogram report for each sample.
   4. We will share the results as a PDF for you to include in your NanoDrop value tables as a column for the average fragment size. Note that the TapeStation also provides information on the amount of degradation or fragmentation of your nucleic acid. In the case of DNA, the value is called the DNA Integrity Number or DIN. The value ranges from 0-10, and for Nanopore sequencing, we strive for higher values >6 to obtain longer reads. Sometimes, fragmentation is inevitable.
8. We will use the sensitive Qubit to determine an accurate concentration of your DNA. While the NanoDrop uses spectrophotometry and various wavelengths to calculate DNA concentration and potential purity, the Qubit uses reagents that specifically bind to double-stranded DNA (in our case, if we are using the High Sensitivity or Broad Range DNA kits).
   1. Take Qubit tubes, one for each sample.
   2. Obtain a tube with the working solution: buffer with a dye that will bind double-stranded DNA and fluoresce.
   3. For each sample, add 2 μl of sample and 198 μl of working solution.
   4. Mix briefly with the vortex and wait 2 minutes, protecting the samples from light.
   5. Use the Qubit to analyze your samples. Your instructors have already calibrated the instrument with two standards.
   6. Record the Qubit values for each of your samples. Note that the DNA concentration is often lower than those obtained by the NanoDrop. We will use the Qubit values for our calculations as we prepare our samples for sequencing. Add your Qubit values for each sample to the table of DNA concentrations.
   7. After reading all samples and recording the DNA concentrations, dispose of the tubes in biohazard waste.
   8. Clean your bench and pipettors with ethanol and tidy up your workplace.

Criteria for Success

We are using real worm samples! We will describe how they were collected and preserved for you to take notes. The amount of DNA obtained will vary depending on the sample type, the efficiency of cell lysis, and the removal of contaminants, for example. Thus, your concentrations may vary. Quality may also vary. Do not fear: you will be able to sequence samples, as we have backups and are working in teams to ensure everyone has an opportunity to prepare DNA for sequencing!

Check Your Knowledge and Practice!