Question

For part 1 of this lab) I collected a soil sample from my campus Part 2) Tested bacteria initial viability Part 3) DNA extraction Part 4) DNA quantification by Nanodrop Part 5) Sample sequencing Part 6) PCR amplification Part 7) Gel electrophoresis Part 8) DNA sequence data analysis (sent sample to another lab)

Directions for Part 3 DNA extraction are in the attached image

QUESTIONS REGARDING PART 3 (DNA extraction)

1) What type of conclusions can be made from initially culturing on nutrient agar (e.g., qualitative assessment, quantitative assessment, preliminary, estimate, descriptive, or bacterial diversity)?

2) What is the purpose of the ethanol used during the DNA extraction?

3) Our primers target the 16S rRNA gene, are there other genes used for metagenomic sequencing?

QUESTIONS REGARDING OVERALL EXPERIMENT

1) What can be a hypothesis for this experiment?

2) What are the controls for this experiment? What are key environmental data to collect that cannot be controlled between samples?

udspring2020 AP%20(7) pdf Part 1: Experiment Setup 1. Collect -80-90 grams of topsoil into your container (up to -100mL mark for red screw top specimen cups). Use a plastic spoon to "shovel soil. Collect exposed soil avoiding roots. twigs, stones, and other debris. Label the container with your group number/identifier, date, and lab instructor's name. Part 2: Bacteria Initial Viability 1. Using the lid of a sterile 1.7 ml microcentrifuge tube, transfer 2 lid full of soil from the container into a test tube with 9mL of sterile water & appropriately labeled, Flick tube vigorously to mix. The soil will settle to the bottom. Perform a 1:10 dilution: Transfer 1 ml of "dirty" water from the first test tube to a second test tube of 9 ml of sterile water (labeled 1:10). Flick tube vigorously to mix. 2. Spread plate 100 ul (0.1 ml) from your 2 tubes (undiluted and 1:10) on to a labeled nutrient agar plate. 3. (optional, if R2A plates are available) Spread plate 100 ul (0.1 ml) from your 2 tubes (undiluted and 1:10) on to a labeled R2A agar plate (for slow-growing bacteria). Part 3: DNA Extraction 1. Using the lid of a sterile 1.7 ml microcentrifuge tube, transfer 2 lid full of soil from container into a labeled Power Soil powerbead tube. Gently tighten cap on the powerbead tube. This is step 1 of the DNA extraction protocol.

Answer 1

Questions regarding part 3 (DNA extraction)

Question 2: What is the purpose of the ethanol used during the DNA extraction?

Answer: Ethanol is used in the final steps of DNA extraction to precipitate the DNA and also for desalting. DNA is negative charged molecule (Polar) and hence it is easily dissolved in water. To precipitate the DNA, salts such as sodium acetate are added to the DNA aqueous solutions. The Na+ ions of the sodium acetate interact with phosphate groups (PO₃) of DNA and makes its less soluble in water. However, water has high dielectric constant that does not allow this interaction. Ethanol has less dielectric constant than water and addition of ethanol promotes Na⁺ and PO₃^- interaction and make the DNA molecule less hydrophilic, leading to its precipitation.

Question 3: Our primers target the 16S rRNA gene, are there other genes used for metagenomic sequencing ?

Answer: 16S rRNA gene are mostly commonly used in metagenomic studies. Other genes such as internal transcribed space regions and 23srRNA gene are also used in metagenomic studies. With advance in sequencing technologies, short-gun metagenome sequencing method sequence whole genome of the species in a community and provide more valuable information than amplicon based sequencing methods such as 16S rRNA and internal transcribed space regions sequencing.