liamharvey's blog

I spent a considerable amount of time yesterday going from point to point. In the morning I drove to parking lot 12; it took about 15 minutes. I then had to walk to the ISB for my Bio383 lab which took another 15 minutes. After lab I walked to blue wall. I then walked to the rec. center. Both walks were no more than 5 minutes. After the gym I walked back to my car in lot 12 and drove home; about 20 more minutes of travel. Later that night, I was picked up by a friend to go to their house, which was about a 10-minute drive. We then went out to the bars which took another 10 minutes. At the end of the night we took Uber which took at least 20 minutes as there were several stops. In total, about one hour and forty minutes of my day was spent getting from point A to point B, a huge chunk of the day.

The results for the agrose gel electrophoresis showed a successful DNA extraction and purification. In RNase treatments there were no RNA bands as should be the case. In comparison, the DNA only samples showed RNA present as expected. The DNA ran to the end of the gel as expected. However, the Nanodrop results did not agree with the gel electrophoresis results. It is unclear why there were difference between the two quantification methods. Perhaps there were errors in the Nanodrop quantification.  

The molecular absorbance spectroscopy via Nanodrop showed very different results. In regard to concentration, DNA1, DNA1 with RNase and DNA2 with RNase all showed concentrations around 150ng/µL. However, DNA2 showed concentration of only 91.3ng/µL. This low concentration could be due to errors when making dilutions or perhaps when using the Nanodrop itself. DNA1 and DNA2 had 260/280 values of 2.07 and 2.04 respectively. 260/280 values over ~1.8 suggest that there is a high concentration of RNA in the sample. For DNA1 with RNase and DNA2 with RNase, values for 260/280 were recorded at 2.95 and 3.45. These results would suggest that the two RNase treatments would have more RNA present than the original samples. This result does not agree with the gel electrophoresis results. In the gel electrophoresis it was clear that the RNase treatments did not have RNA present. Perhaps the presence of RNase interfered with the Nanodrop, or there were issues with the computer. DNA1 and DNA2 had 260/230 values of 1.65 and 1.51. 260/230 values below ~2.0-2.2 suggest that carb carryover occurred. Perhaps not all of the carbohydrates which should have been removed via KAOc were indeed removed. DNA1 with RNase and DNA2 with RNase had 260/230 values of -1.43 and -0.73. These values are under the ~2.0-2.2 range, however, they are negative which could mean something else has occurred. The 260/230 value is a ratio, so a negative value may indicate something other than carb carryover. The negative values could also be another possible error with the Nanodrop.

Lanes three and five of the agrose gel electrophoresis show three bands in each lane. The band at the top of the gel is the DNA, which is electrically charged and ran up to the positive end of the gel. The two bans lower in the lanes show RNA present in the sample. In lanes four and six there is one band at the top of the gel but no bands below. The band at the top is again the DNA which ran to the positively charged end of the gel. These two lanes were treated with RNase, which explains why there are no other bands in the gel. The RNase enzyme appears to have successfully broken down the RNA in the treatment samples.

Two separate DNA samples were made in this process. Each sample was then quantified using two different methods: molecular absorbance spectroscopy using a Nanodrop, and 0.9% Agrose gel electrophoresis. Along with the two DNA samples, a RNase treatment was made and quantified from each DNA sample. The RNase treatments were then compared to their respective DNA samples with both quantification methods.

After DNA had been extracted and degradation prevented, the DNA sample had to be purified. Protens, carbohydrates, and lipids were all present in solution and had to be removed for a pure DNA sample. Because carbohydrates and proteins are insoluble in solutions containing high concentration of potassium acetate, KOAc was used to separate these macromolecules. The macromolecules formed a pellet when in KOAc solution which was then removed. To rid the solution of lipids, an alcohol solution of isopropanol was used which formed the DNA into a pellet separate from the lipids. The pellet was then separated from the lipid solution and rinsed with EtOH.

The purpose of this experiment was to extract, purify and quantify the DNA of Brachypodium distachyon a common grass species. The extraction and purification process involved three steps; breaking up tissue and cells, preventing DNA degradation and removing unwanted molecules. To break up the tissue and cells of B. distachyon, mechanical grinding was first performed using a ball mill to break up the extracellular matrix. Chemical disruption was performed next using the detergent sodium dodecyl sulfate (SDS) with metal chelation using ethylene diamine tetra-acetic acid (EDTA). The final step in breaking up tissues and cells was heat disruption to release the DNA which was done on a hot bock set to 65°C. After DNA had been extracted from B. distachyon tissue, it was essential to prevent the enzyme DNase from degrading the free DNA. Preventing DNase from breaking down the extracted DNA was done with the already added EDTA. DNase requires Mg⁺⁺ as a cofactor; by using EDTA to chelate with Mg⁺⁺ prevents DNase from acting.

DNA or deoxyribose nucleic acid is the unit of heredity that almost all organisms on earth have and use as a carrier of genetic information. DNA forms DNA forms an alpha helix structure with bae pairing of the purine bases adenine and guanine with their complementary pyrimidine bases thymine and cytosine. DNA is transcribed into RNA which is then translated into proteins which can then perform an array of important functions in living organisms. DNA is complex and essential to the functions of nearly all living organisms. It’s complexity and importance in our understanding of how we function are what make it important to continue studying.

The organism is about 26mm in total length. This organism has a spherical body and long, thin tail. The tail length is about 15mm and the body length is about 11mm. It's body is about 3mm wide. The organism is a tan color but is see-through. Inside the body are visible and symmetric organs, some of which are white and some are dark. The white organs appear to make a figure-eight structure around the darker organs.  The organism moves similar to a caterpillar in that it perorms an abduction/adduction movement. At first the organism did not move. After some time, however, it began to wriggle around. The movement of the organism seems to be random, with no path or direction. It often flips over and rolls around. The eyes are not visible; perhaps the tail functions for environment sensation. The tail is longer than the body by about 5mm. It does not appear to have any appendages. There are however, small bumps on its underside which may be functional is movement or traction. Based on it's random movement it is likely that the organism is in a juvenile stage. 

The organism is about 26mm in length. It has a spherical body and long, thin tail. The tail length is about 15mm and the body length is about 11mm. It's body is about 3mm wide. The organism is a tan-ish color but see-through. Inside the body are visible and symmetric organs, some of which are white and some are dark. The organism moves simalr to a caterpillar in that is does an abduction/adduction movement to move around. At first, the organism did not move but after some time it began to wriggle around. The movement of the organism seems to be random, with no path or direction. It often flips over and rolls around. Eyes are not visible, perhaps the tail functions for environment sensation. The tail is longer than the body. It does not appear to have any appendages. There are however, small bumps on its underside which may be functional is movement/traction. Based on it's random movement I would guess that the organism is in a juvenile stage.