Week Two: (June 28 - July 2, 2010) Computers and DNA

Hello again!  I have learned a lot this week and I feel like my research is really getting underway here at CMOP.

6/28/10: I ran a PCR and gel by myself for the first time and obtained accurate results.  Afterwards, Dr. Herfort told me I will be learning how to clone samples sometime next week.  In light of this, she had me label petri dishes and pour agar into them with Sheedra.  Once we finished we went over what I would be doing the next day and starting packing supplies for the Oceanography Camp boating trip.

6/29/10: Dr. Herfort was gone today with the Oceanography Camp, so she had me run a program called MOTHUR to analyze sequence sets of DNA code to identify species in bacteria, as well as species relation in M. rubra.  In order to use MOTHUR I have to use a program called BioEdit to isolate sequences, but it doesn’t work on my Mac, so I had to use the lab PC.  After running the program for rarefaction to give me species relation I opened up the .phylip.fn.rarefaction file in Excel and made an xy-scatter plot of the data.  The only down side of this job was that there were 17 sequences and I could only process one at a time, so on Thursday I will try another program command to do all at once.

6/30/10:  Today several ASE Interns and Oceanography Camp students went out sampling in Ilwaco Bay and the surrounding area.  In order to sample and collect salinity and temperature data, we lowered the sampling device in one meter increments until we reached 12 meters, then raised the sampling device up to one meter and collected a water sample.  We then sample rinsed (decontaminated) our collection bottles, filled them to 400 mL with site water, and filtered the water through a 0.22 micron Sterivex filter to collect microorganisms and DNA/RNA.  To preserve any DNA/RNA not in a cell, we added RNAlater and sealed the Sterivex.  At our second sampling site the two groups switched roles, one taking the samples and one recording the data at each meter.  It was a fun day, but I most likely will not be processing the results tomorrow because Dr. Herfort wants me to run the computer program, MOTHUR.

7/1/10: Today I was learning how to do a rarefaction for all 17 of our sample sets using MOTHUR.  Even though there was a manual on the website for this “shared rarefaction” (the command name), the results were not the same as the individual rarefactions.  After talking with Dr. Herfort about the results we decided to import all of the individual sequence set data into one excel file and create an x-y scatter plot. 
The Oceanography Camp students were in the lab today, so I helped Dr. Herfort and Pete set up for them.  Even though I was busy working on the computer, I would occasionally glance over and see the students having a lot of fun, bringing back memories from my time at the camp last year.

7/2/10:  Today Dr. Herfort taught me how to extract DNA/RNA so I could process samples taken on the May cruise at the coast.  Most of the work done in DNA/RNA extraction is preparing your lab bench because no RNAse, a substance that degrades RNA, can be present.  Once the bench is clean, you have to remove the filter from the Sterivex and start the extraction.  Dr. Herfort explained the extraction process to me like this:  you have the first extraction to isolate the microorganisms, then the second extraction to isolate the DNA/RNA, then purification, then storage in a -80 °C freezer to preserve the sample DNA/RNA.  While there are more steps involved, this is the basic premise of DNA/RNA extraction.  I think I will be doing this for at least two weeks, because I have 16 samples and can process roughly two per day.  I really look forward to perfecting my techniques for this process.

Until next time,

~ Deirdre