Dan's+Notebook

Daniel Bascom October 9, 2011 FISH 441 Lab 1 Steven Roberts


 * Summary**

The purpose of this lab was to introduce the basic methods and principles behind RNA and protein extraction from a tissue sample. Samples were taken from a Pacific oyster and prepared for further study using a Bradford Assay.


 * Materials and Methods**

//RNA Extraction// For the RNA extraction, we took a prepared gill sample from those provided, recorded the weight, then placed the sample in a 1.5mL snap-cap tube, labeled with our initials, date, and contents, with 500uL of TriReagent. We then homogenized the sample by grinding it within the tube with small disposable pestel, this releases any DNA, protein, and RNA into the TriReagent so as to isolate the RNA from the rest of the tissue for further analysis. We then added another 500uL of TriReagent and vortexed the sample to further mix its contents, and placed it on ice.

Time started(sample removed from ice): 13:55 Time finished(treated sample placed back on ice):14:10 materials used: 1.5mL snap cap, 1000uL pipette, disposable pestel, 1000uL of TriReagent, tweezers tube labels: DBB, 10/4, gill

//Protein Extraction// For the protein extraction, we used a prepared mantle sample. After recording the weight, we placed it into a labeled 1.5mL snap-cap tube and added 500uL of CellLytic MT. We then homogenized the sample with a new disposable pestel, then mixed the contents by repeatedly inverting the tube. Next, we and 3 other groups placed our samples into the refrigerated microfuge for 10 minutes. During this time we labeled and prepared our tubes and cuvettes for the next phase.

After coming out of the microfuge, we extracted the separated proteins ( dissolved RNA user:emmats) from the top of the tube, and placed it into the prepared tube, 15uL of this was taken and placed into a separate tube for the Bradford Assay. The Bradford sample was diluted to a 1:2 ratio by adding 15uL of water bring the total volume of the container to 30uL. A second tube was filled with 30uL of water to serve as a blank/calibrator for the spectrohometer. 1.5mL of the Bradford reagent was added to both tubes and mixed with the pipette and by inverting the tube.

1000uL was taken from the blank sample and placed into a disposable cuvette. After cleaning with a kimwipe, this was then used to calibrate the spectrophometer. In a new disposable cuvette, 1000uL of our protein was deposited and its absorbance measured using the calibrated spectrophometer. The cuvette was removed, stired via pipette, and then remeasured. Our two values recorded came out to 0.347 and 0.348nm, with a mean value of 0.3475nm. Based on the graph provided, with a absorbance to concentration ratio slope of y=101.9x, the concentration of our protein was 5835.4 ug/mL What was the wavelength at which you measured absorbance? user:emmats

y=1013.9(.03475)=2917.7x2 for dilution factor->5835.4

Time started-14:22, Time finished 14:45 materials, 1000ul pipette, 100uL pipette, snap cap tube, disposable cuvettes x 2, disposable pestel, kimwipes

tube labels: DBB, 10/4, Protein, BA; blank

I was expecting a much larger sample necessary for running an assay and for protein extraction. I expect our results were similar to the rest of the classes, however results will most likely vary depending of the tissue samples used. The next step will to be to run the actual assay and measure the gene expression within each tissue.
 * Conclusions**

This lab taught me, and the rest of our class, the techniques and principle behind the isolation of different cellular components, in this case RNA and protein. It this instance, we will complete our measurements of gene expression in the next lab. I believe we will see significant differences between tissues in both protein concentration and RNA expression given the different cellular structures present within each type of cell. These methods can be used to study how an organism responds to almost any stressor be it changes in pH, temperature, or other changes in the environment such as predation or food availability. They could also be used to determine the life stage of the organism. I would like to know more about the Bradford assay and the differences among different assays and other forms of genetic analysis, I also wish I knew more about shellfish biology so as to better understand differences among the tissues used, and how those differences will effects our measurements.
 * Reflection**