Craig+Banta's+Fish+441+lab+notebook

10/29/2013

This lab was just putting the finishing touches on the layout of our research projects and starting them. We discussed using E. coli and Vibrio and we are going to go with whichever population turns out better. Then, we also decided to add heat to the experiment along with the UV rays.

Then we got our cultures started by spreading E. coli and Vibrio on an LB medium to grow in population. We will use these cultures to grow in LB broth on Monday.

We also figured out primers for our respective genes for Vibrio. And that was about it for lab this week.

10/22/2013

Summary: The purpose of this lab was to do a first take of analyzing a conventional PCR by use of agarose gel and to perform a Western blot. The particular samples I worked with were two separate tissue samples from the mantle of a Pacific oyster for both series of testings.

Methods: Western blot Add 15 microliters of protein stock and 15 micro liters of sample buffer to a screw cap tube and boil mixture for 5 minutes. While they were boiling, a gel box and gels were assembled for the proteins to be placed in. Once loaded, the samples had an electrical current run through them (150V) for 45 minutes. The gels were then removed from the box and transferred onto the western blot machine. The machine was run for 30 minutes at 20V and then the gels were removed and washed with pure water. The membranes were placed in a plastic box with 10 mL of blocking solution, covered, and incubated for **30 minutes.** The rest was left for the TA.

Electrophoresis procedure The agarose gel was already made before class by Claire. The gel was placed in a gel box and covered with a buffer solution. A 100bp ladder was added in the far left lane and 20 microliters of our PCR samples were added into the remaining slots in the gel. The gel was then run at 100V for about an hour.

Results [|Western blot 1] [|Agarose gel 1]

Conclusions Nothing was out of the ordinary. Everything looked about as I expected it to. I was in well 9, so there was only one line for both the gel and the blot, so there were no other genetic sequences in my samples.

Reflections:

This was more practice for the individual projects we will be doing in the upcoming weeks. It was practice for using both an agarose gel to analyze a PCR sample and a Western blot technique to analyze a protein stock.

10/15/2013

Summary: This lab was again more preparation for the project later in the quarter. It was practice in the procedures for qPCR and analyzing the qPCR by computer data. We extracted cDNA by reverse transcribing RNA from a new sample of oyster tissue.

The other part of this lab involved extracting proteins from oyster tissues. It was extracted from a tissue sample from the mantle of a pacific oyster.

Methods:

First, a master mix was created using 62.5 microliters of SsoFast EvaGreen supermix, 2.5 microliters of upstream primer, 2.5 downstream primer, and 52.5 microliters of ultra pure water. 24 microliters of this mix was added to 4 small test tubes, with 2 being the control and 2 containing the cDNA. The 4 tubes were then put on a tray and run through a machine to analyze the cDNA.

For the protein extraction, 500 microliters of CellLytic MT solution was added to the tissue sample and homogenized. The tube was then spun in a refridgerated microfuge for 10 minutes.

10/1/2013

Summary: The purpose of this lab was to practice separating DNA, RNA, and proteins from different tissue samples and to prepare an RNA sample for next week's lab. This was done using DNazol to extract the DNA and TriReagent to separate the RNA. The samples I worked with was a sample of gill tissue from a Pacific Oyster and a sample of mantle tissue from an Olympic Oyster.

Methods:

We were given two different tissue samples, one to extract RNA from and one to extract DNA from. For the RNA, 0.5 mL of TriReagent was added to the tissue sample and then homogenized with the tissue. Then, another 0.5 mL of TriReagent is added to the solution and it is stirred in the vortex for about 15 seconds. Then, this solution is stored at -80 degrees Celsius for next week's lab.

For the DNA, we added 0.5 mL of DNazol to the tissue sample and mixed until it was homogenized. It sat for 5 minutes at room temperature, then spun for 10 minutes. The DNA sample was then washed with 100% ethanol and then 75% ethanol. Then, the DNA sample is dissolved in DEPC water and measured in a Nanodrop.

There were no deviations from the method, aside from a couple extra minutes of the DNA sample waiting at room temperature before spinning in the centrifuge.

Results: DNA concentration: (ng/µL) = 266.0 260/280 = 1.82 260/230 = 1.37

Conclusions: The 260/280 concentration is within a reasonable range, but the 260/230 concentration seems a bit low, so there could be some contaminants in the sample.

Reflection: This lab is a practice to get used to the procedure of separating and measuring DNA and RNA from tissue samples for a later project. In this particular lab, TriReagent was used to separate RNA from the tissue sample. And DNazol was used to isolate the DNA from the tissue sample and a Nanodrop was used to measure the concentration of the solution. By isolating the DNA and RNA, they can be used in PCR and qPCR to check for particular sequences and the nanodrop can be used to test the purity of the DNA and RNA strands.

This lab was pretty short and straightforward in terms of what we needed to do to get our samples, but it could have been clearer on why we got them in the first place. I had to look up the reasons online to double check why we wanted to test the samples like we did. So, I would like to know if my reasons for doing this are not correct just in case I am misinformed on the matter.