6 October 2013
Proteins of Interest for Lab Project

I have been having a hard time finding proteins I would want to use for my lab project but I have come up with one possibility. The protein Nacrein is involved in the calcification of the mollusk Pinctada fucata. It is a calcium concentrator and is involved in the production of carbonate. My interests lay with Foraminifera however, and I would like to find a protein involved in calcium carbonate secretion in the Elphidium or Bucella genera, but this will take a bit more time to find.

1 October 2013
Lab 1: DNA and RNA Isolation.

Summary of lab:
The purpose of today's lab was to familiarize and practice techniques for DNA and RNA isolation from sample tissues. The processes of RNA isolation was begun but not finished. RNA isolation is accomplished in this lab with the use of TriReagent which uses guanidine isothiocyanate, phenol and pH to seperate RNA from other cellular components of whole tissue. The processes of DNA was completed and a quantification of DNA from tissue was achieved. DNazol was used to separate the DNA from other cellular components of whole tissue by precipitating DNA from the cell.

Material and Methods:
The lab began by starting the process of RNA isolation. A sample of 75mg of mantle tissue from an Olympia oyster was obtained and kept on ice at all times that it was not actively being manipulated. Five hundred microlitres of TriReagent were added to the whole tissue and the tissue was then homogenized with a sterile pestle. The sample was then labeled as RNA with my initials and stored in the freezer by the TA.
At this point DNA extraction and isolation procedures were initiated. A sample of 38mg of gill tissue from a Pacific oyster was obtained and kept on ice at all time that it was not actively being manipulated. The procedure began by adding 500 microlitres of DNazol to the sample and homogenizing it. This processes was repeated before the sample was allowed to incubate for approximately five minutes. After the incubation period the sample was spun at 10.000 x g for ten minutes at room temperature. After this, the supernatant was transferred to a sterile tube and 500 microlitres of 100% ethanol was used to wash the sample. The sample with the ethanol was inverted eight times and left at room temperature for approximately one minute to allow the precipitate to form and settle at the bottom of the tube. The ethanol wash was removed from the tube and this processes was repeated twice more using a 75% ethanol wash. Once the precipitate had been washed and as much ethanol as possible was removed from the sample 300 microlitres of a 0.1% DEPC water was added to the sample. The sample was dissolved by agitation by pipetting several times. In the process of this agitation two drops of the sample were splashed onto the table and were lost due to cross contamination issues. This concluded the DNA isolation protocol.
After the DNA had been isolated it was transferred to a spectrometer in another lab and 2 microlitres were used on the spectrometer to attain quantification of the DNA sample. The DNA was then labeled with DNA, my initials and the date then given to the TA.

The quantification of the DNA gave a concentration of 14.6 miligrams per microlitre, an A260/A280 ratio of 2.09 and an A260/A230 ratio of 0.06. The expected range of values for the A260/A280 for good quality DNA is between 1.7 and 1.9.

According to Wikipedia(1) a value of 2.00 in an A260/A280 ratio indicates 100% DNA extraction.In light of this an A260/A280 ratio of 2.09 does not make sense. It is possible that these findings were contaminated due to improper cleaning of the spectrometer or because of the calibration error that occurred during a quantification two tests before this samples.

DNA is an integral part of this classes study of the relationship between external and internal enviroments in aquatic life. Outside stresses can have an impact on the internal physiology of an animal because those stresses can affect how and which genes are expressed in an animal. DNA, RNA and proteins are the genes and the mechanisms by which the genes "purpose" is fulfilled. If these important pieces of the cell can be measured, the physiological impacts of the environment on the animal can be measured and used to understand the relationship between environment and physiology, which is the ultimate goal. This particular lab is used to practice DNA and RNA isolation which is an important tool in the study of DNA and RNA. The measurement of concentration of DNA that was obtained in this lab can be used as a guide to how successful a student is in attaining the desired results in this particular DNA isolation protocol. This protocol can be used in any study where the DNA of a tissue must be isolated and studied. This could include studies where the entire genome is read and archived, studies where the DNA of individuals is compared, or DNA changes in respect to time in one individual are compared. As a paleontology student with a strong background in geology and weak one in biology, much of this protocol is quite new to me. However, the importance of the procedure and the methods of the protocol are much clearer to me now. I do not know much about the specifics of how the reagents we used broke the cells down into their respective parts and isolated the DNA, but I do know that this protocol used methods of both mechanical (through the use of the pestle and centrifuge) and chemical (through use of the various reagents and solutions) breakdown of the cell before isolation via DNA precipitation was used. My only regret is that I do not have a strong a frame of reference in regards to the processes in this lab. I have little conceptual background to draw conclusions from and as such I do not have an intuition about my findings.

(1) Wikipedia Necleic-acid quantitation page: http://en.wikipedia.org/wiki/Nucleic_acid_quantitation#Protein_contamination_and_the_260:280_ratio. Retrieved 10/2/2013