aspark's blog

The unknown DNA sequence was saved as “AHP - Gene Sequence” as a text file on Microsoft Word and translated using the Bioline Six-Frame Translation website with the following settings: Output: 20 amino acids in one line, code: one letter, frame number: all. The whole map under “Sequences alignment” was copied, pasted into a new document, and saved as “AHP, Angela Park - Working Map File” in Microsoft word format, changing the font to 10 point Courier and the side margins to 0.7”. In order to obtain predictions of its structure, protein sequence, and coding sequence, the unknown sequence was then ran in the FGENESH website, selecting “Brachypodium distachyon” as the organism and choosing “print exon sequences for predicted genes” in “Advanced options” to get sequences of all the predicted exons separately. The diagram under “Show picture of predicted genes in a pdf file” was saved as “FGENESH Prediction Diagram - AHP.” The protein and coding sequences were then separately saved into new text files as “FGENESH Protein Sequence Prediction - AHP” and “FGENESH CDS Prediction - AHP” respectively.  

Another possible experiment our class could perform is a study on when different species flower. April is the month that flowers begin to sprout and flower again, and we can measure the difference between different species at this time. Individuals could find random plants and record the species, color, size, shape, and location of the flower over time. In this way, we can track the level and speed of development of various species. We could also study plants that we grow ourselves. Everyone in the class could grow different flowers, or we could choose a few select species to divide amongst the class. We would subject these plants to varying conditions while growing them, including differences in lighting, temperature, watering, soil, and density of flowers grown in an area. This study could reveal information on the best conditions for flower growth and development and at what point the conditions are not enough to sustain correct flowering. 

After performing agarose gel electrophoresis on DNA samples, the samples treated with RNase were easily distinguishable. Samples treated with RNase displayed a single band of DNA, while untreated samples displayed the same band along with a smear where the much smaller RNA strands were deposited. From this I concluded that the RNase successfully eliminated the RNA contained within the extraction samples.

For the sample treated with RNase, the ratio of the absorbance at 260 nm to the absorbance at 280 nm was 1.26, which is significantly lower than the 1.8 ratio that indicates pure DNA.  From this I concluded that the DNA extraction product was contaminated. The untreated sample had a higher ratio of 2.08, indicating that the DNA was contaminated with RNA, which has a larger ratio than pure DNA.

Additionally, the concentration of the RNase treated DNA was 0.2590 µg/µL. Because the final volume of the extraction was 50 µL, I calculated that 12.95 µg of DNA extracted. The concentration of the untreated DNA was 200.7 ng/µL, which was unexpectedly low since we would expect the nucleic acid concentration to be higher before RNA is eliminated.

Another possible study we can do as a class is a study on produce or fruit from different supermarkets. We could all buy the same item from various supermarkets, and we each person could record its color, if it's organic or not, and where it was grown. We can leave the fruit/produce out and observe how it rots over time. We could also measure the size of the food item if we need more variables to study. This experiment would be impactful for learning about preservatives used in the food we purchase at grocery stores. It could also show us the difference in shelf life between organic and non-organic foods. There are various grocery stores students can visit to purchase their sample, including Trader Joe's, Big Y, Stop and Shop, Aldi, Target, and Whole Foods. Possible food items to observe are apples, spinach, bananas, avocados, tomatoes, carrots, etc. If its a food item with multiple varieties, such as apples, we would probably keep the variety observed constant. We may have to do research on shelf life ahead of time to choose a food item that won't rot in too short or too long a time. 

I have multiple ideas for the proposal project coming up, and when deciding which to pursue, there are many factors to consider. Each group within the class needs to be able to research or explore a different aspect of the organism(s) and come to their own conclusion. The topic should be interesting, and it should be something relatively accessible for students at UMass Amherst. Whatever we observe or research also needs to be available early-mid April, which is when we will be conducting the actual project. One idea is to observe the ducks and geese at the UMass pond. Individuals can record the temperature outside, the number of geese and ducks on the water, the number of geese and ducks on land, the number of young and adults, and the time of day. This will give us insight into the duck and geese behavior and maybe be able to make conclusions about the population present on different days and at different times of the day. Another idea is to observe human behavior at bus stops. The control can be that we all observe a single bus stop, perhaps Haigis Mall. We can record the temperature, weather conditions, number of people waiting outside, the clothing of the people waiting, the activity of the people waiting, and so on. Perhaps we can make some conclusions about how students behave depending on outside conditions. 

The 50 µL of extracted DNA in T10E1 was divided evenly between two tubes, and one was treated with 2 µL of RNase A while the other had 2 µL of sterile water added. Both were incubated for 30 minutes at 37°C before being analyzed by a NanoDrop spectrophotometer. The genomic DNA was also measured via agarose gel electrophoresis. A 50-mL tube of 0.9% agarose containing SYBR Safe was taken from a 65°C water bath, poured into a mold, and set aside to set. A ½ dilution of both the treated and untreated genomic DNA samples were created by combining 10 µL of the DNA stock solutions and 10 µL of dilution buffer. The four loading samples to be run in the gel (genomic DNA treated with RNase, ½ dilution of genomic DNA treated with RNase, untreated genomic DNA, and ½ dilution of untreated genomic DNA) were created by combining 10 µL of each DNA stock solution, 5 µL of loading dye, and 9 µL of water. The gel was loaded with 2.5 µL of DNA ladder in the first lane, 5 µL of DNA ladder in the second lane, and 5 µL of each of the four loading samples in the next four lanes, and the gel was run at 100V for 30 minutes. The gel was visualized via blue light.

A 5 cm segment of B. distachyon leaf was ground up into a powder by a Verder ball grinder after being frozen in liquid nitrogen. 600 µL of DNA extraction buffer (100 mM sodium chloride, 50 mM Tris, 25 mM ethylene diamine tetra-acetic acid (EDTA), 1% sodium dodecyl sulfate (SDS), 10 mM beta-mercaptoethanol) was then added and mixed to break up the cells and prevent DNA degradation. The tube was incubated in a 65°C hot block for 10 minutes then iced. Next, 250 µL of 5M potassium acetate was added and mixed, and the tube was incubated on ice for 20 minutes for the carbohydrate and proteins to precipitate. The tube was centrifuged at maximum speed for 10 minutes, and the supernatant containing the DNA was transferred into a new tube containing 600 µL of 100% isopropanol. This solution was mixed, and the DNA was allowed to precipitate before centrifuging the tube at maximum speed for 15 minutes. The supernatant was removed, and the pellet containing the extracted DNA was rinsed with 300 µL of 70% ethanol and centrifuged for another 10 minutes. The liquid was pipetted out, and the remaining ethanol was allowed to dry completely. Lastly, the tube had 50 µL of T10E1 (10 mM Tris, 1 mM EDTA) added to it, which preserves the DNA, and was incubated at 65°C for 10 minutes. The pellet was mixed into the solution by pipetting up and down.

The samples treated with RNase only displayed the band of DNA while the samples untreated with RNase displayed a band of DNA and a band of much smaller RNA further from the wells. . From this we can conclude that the RNA contained within the extraction was eliminated in the RNase-treated samples.

For the sample treated with RNase, the ratio of the absorbance at 260 nm divided by the reading at 280 nm was lower than the expected 1.8 at 1.26, indicating that the DNA extraction was not very pure and may have contained contaminants. The sample not treated with RNase had a ratio of 2.08, which indicates the DNA was contaminated with RNA, which has a larger ratio than pure DNA. The concentration after eliminating RNA with RNase was 0.2590 µg/µL, meaning there was 12.95 µg of DNA present in the original 50 µL extraction solution.

Strangely, the concentration of nucleic acids was 259.0 ng/µL for the DNA treated with RNase, which is higher than the 200.7 ng/µL concentration of DNA untreated with RNase. The concentration is expected to be lower once the RNA is eliminated. This can be due to a unequal dividing of the genomic DNA before treating one sample with RNase.

In order to study the DNA of any organism, the genome must first be extracted for use. DNA extraction involves three crucial steps: tissue and cell disruption, preservation of the DNA, and clearance of extraneous cell components, including carbohydrates, proteins, and lipids. Once DNA has been purified, it is valuable to analyze the DNA sample in order to determine the yield and purity of the extraction product. Often times when DNA is extracted, RNA also remains in the contents since it is a nucleic acid similar to DNA; however samples can be treated with RNase, an enzyme that degrades RNA. DNA analysis can be carried out through gel electrophoresis, and for more information, it can also be spectrophotometrically analyzed. In spectrophotometry, the absorbance of light traveling through a sample is measured to determine the concentration and purity of the contents.

In this lab, we extracted the genomic DNA of Brachypodium distachyon. We then quantified the DNA using gel electrophoresis and absorbance spectrophotometry in order to verify the purification of the DNA, comparing samples treated and untreated with RNase A.

There are two categories of enzymes based on their kinetic features: Michaelis Menten and allosteric enzymes. Michaelis Menten enzymes display a hyperbolic curve when initial reaction rate is plotted against the concentration of the substrate, and they have kinetic parameters that are used to study their function. One parameter is the Km, which is the substrate concentration when the reaction rate is half its maximum. This value indicates how quickly an enzyme reaches maximum activity, and it can be considered a measure of affinity for particular substrates. On the other hand, Kcat is the turnover number that indicates how quickly an enzyme catalyzes a reaction once it’s bound to its substrate. The higher the Kcat, the faster the enzyme changes substrate to product once bound. These two parameters are compared to determine the specificity constant, Kcat/Km, which is the measure of an enzyme's overall catalytic efficiency. It takes into account both substrate binding and speed of product formation, so even if an enzyme's Kcat is low, if its Km is low, it can still be considered relatively efficient.