Lab questions for Environmental Drivers of Symbiosis (NEED HELP PLEASE) PROTOCOL
ID: 218288 • Letter: L
Question
Lab questions for Environmental Drivers of Symbiosis (NEED HELP PLEASE)
PROTOCOL: In this laboratory, students seek to determine the response of anemones to each of the four following conditions: high light/high temperature, high light/regular temperature, low light/high temperature, and low light/regular temperature. Students will use genetically identical clones of the same anemone. These anemones are hosting different symbiont species: Symbiodinium linuchaea, S. minutum and S. trenchii. By using clones, the results obtained will be indicative of the presence of specific symbiont species rather than differences between anemones. Using qPCR, students will quantify the presence of Symbiodinium (quantifying the presence of the gene coI) in each host species (quantifying the presence of the gene 18S). Each section will work with only one holobiont, but all four environmental treatments. At the end of the experiment, sections will share and compare data.
In this laboratory, students will quantify the amount of protein present in their sample by using Bradford assay.
Using a spectrophotometer to compare against solutions of known protein concentrations (standards), students can calculate the protein concentration of their samples. A standard curve for this assay relates the concentration of protein in solution with the absorbance of that protein following reaction with Bradford reagent (Figure 1). You will use the equation on this graph’s linear regression (trendline) in order to calculate your sample’s protein concentration.
1.- If sample A reaches a fluorescence level of "12" after 15 cycles of PCR for a symbiont gene and sample B reaches a fluorescence level of "12" after 18 cycles, which sample has more symbionts? By how much?
2.- Why was it important to use chelex in the DNA extraction? What is the role of magnesium in terms of DNA?
3.- Imagine that S. minutum provides its host with more amino acids than S. trenchii, so S. minutum hosting anemones have more protein per cell. How does this affect the validity of symbiont density as measured as symbionts/host protein? How about for symbiont density measured through the symbiont CO1/host 18S approach?
4.- Why is it important to normalize symbiont values to a similar measurement of the host? Give an example of a scenario where not normalizing symbiont measurements could produce misleading results.
Explanation / Answer
1) In real-time PCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle. So if we reach the same level of fluorescence at different cycles, this means we can infer we have more PCR product at the lesser cycle. In this case, sample A seems to have more symbionts than sample B. Each cycle grows exponentially, so we can infer 3 fold difference.
2)It is important to use chelex in order to get cations out of the medium, this resin binds to them. This include the cation magnesium, which is important because it is an essential cofactor for DNases. This enzymes could degrade our DNA samples.
3) If we measure the proteins (Bradford assay), then we could overestimate the symbiont density, since more protein will not correlate with more population density per cell.
Symbiont density measured through qPCR (symbiont CO1/host 18S) approach will not have this problem. Since we are counting copies of genes in both species (host and symbiont).
4) It is always important to normalize values in order to compare results later. Imagine we only compare the different symbionts values and different values per host. How can we compare their density if we are using different measures for symbionts and hosts? We need to make it comparable.
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