Phone Number: (240) 751-2833
July 20, 2010
Dissected Strongylocentrotus purpuratus using procedures derived from Wallace and Taylor (2003). Procedures went as follows:
1) Following the suggestion of Wallace and Taylor, we (myself and Marie LaRiviere) removed the urchin's spines in the plane equidistant between the aboral and oral sides, the region that Wallace and Taylor (2003) referred to as the equator of the urchin. Under Dr. Harvell's advice, we removed these spines using vice pliers that were tightened to the maximum degree.
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| "lane 1: ladder; lane 2: neg control; lane 3: neg con; lane 4: 3; lane 5: 2; lane 6: 1;, lane 7: control, lane 8: spx; lane 9: Aspergillus; lane 10: neg con; lane 11: neg con; lane 12: ladder" (image and caption from the lab's main page) |
"lane 1: ladder; lane 2: neg control; lane 3: neg con; lane 4: 3; lane 5: 2; lane 6: 1;, lane 7: control, lane 8: spx; lane 9: Aspergillus; lane 10: neg con; lane 11: neg con; lane 12: ladder" (image and caption taken from the lab's main page)
The gonads can be distinguished from other organs due to its placement along the test and its yellow color. The ampellae occupy a similar location but are white in color. The features of the digestive system can be disguished from other systems as it begins within the lantern of Aristotle on the oral side and then snakes around the test, occupying the majority of space, to terminate at the anus on the aboral side. The water vascular system could easily be mistaken for a part of the digestive system, as it begins in the ring canal, which encircles the lantern of Aristotle and terminates at the madreporite, which is near the anus. Care can be taken, however, to distinguish the lantern of Aristotle from the stone canal, thus distinguishing the water vascular system from the digestive system, at the oral end. Also, the digestive system has a brown color while the stone canal (the region of the water vascular system that connects the ring canal to the madreporite) is white or transparent.
Other members of the class also performed dissections on oysters, geoducks, gastropods, sea cucumbers, sea stars, and anemones. These dissections will assuredly be described with greater detail in other students' lab notebooks.
July 21, 2010
Compared histological slides of healthy tissue from the red abalone Haliotis rufescens with tissue infected with a microsporidian, as well as other molluscs that were displaced by Dr. Friedman. Some of the key observations made in this section of lab include:
-Gill tissue can be differentiated from other tissues by its thin, snaking appearance.
-Hemacytes can be identified as small, dark dots in the tissue.
-Healthy hypobranchial gland tissue can be identified by its pink coloration under H&E staining (such tissue is called esophillic), its broad, wavy appearance, and its vicinity to the mantle.
-Ova can be differentiated from other cells as they are large and circular, more so than other types of cells. They also tend to be found with other ova.
-Sperm can be distinguished because it occurs in circular pockets. The density of sperm is greatest in the center of the pocket and decreases as one moves toward the circumference.
-Nerve cells can be distinguished by their solid, homogenous color. They should be esophillic.
-The infected sample had a region of tissue with lots of little brown circles. This may be a result of the infection.
In addition, we also collected roughly 66 Littorina sitkana and 66 L. scutulata from Cattle Point, Washington, USA, and dissected them to to determine whether they were infected with larval trematodes. The protocol for dissection that I used varied through the process as I adopted new practices and abandoned old ones. The protocol that I feel worked the best went as follows:
-Crack the shell of the snail with vice pliers. In the past I have also used rocks and hammers to crack snail shells and have generally been pleased with the results. Either procedure works well.
-Place the snail, with the cracked shell into petri dish.
-Rinse the snail with seawater (henceforth referred to as wash water), then remove the soft parts of the snail from the shell using forceps under a dissecting scope. In my experience, the key to a clean removal (one where the gonads or digestive glands do not get torn off) is to i) make sure that the shell does have some damage near the apex of the spire and ii) be sure to sever the columellar muscle from the shell. The latter step can easily be done with fine tipped forceps. Sometimes it also helps to press the operculum with a pair of forceps through the aperture until the soft parts slip through a hole in the shell. This allows you to tear off the soft parts without severing tissue per se.
-Examine the broken shell material and wash water for cercaria using a dissection scope.
-Examine the digestive gland/gonad region of the snail for larval trematodes. At least in my observation, they will appear white and can be easily seen at ~150% magnification, although I imagine this may differ with species. Tearing the digestive glands and gonads up using forceps may be necessary (Harvell, personal communication; Blakeslee, personal communication).
I dissected three L. sitkana and five L. scutulata. Of these, one L. sitkana was infected with both rediae and cercariae. A brief description can be found on the class data sheet and a photograph at 140% magnification (I believe) was taken of a cercaria. The photo is stored on the computer in Lab 5 under the file name KJS8_cercaria. Tissue samples were also taken for DNA and RNA extractions of snail tissue. The DNA tissue sample was all of the snail soft parts except the digestive glands, gonads, head, and operculum. The RNA sample was a subset of the DNA sample, in my case coming from the foot tissues. DNA samples were refrigerated, and RNA samples were stored on dry ice.
July 21, 2010
Today we examined a sample of five Armenia sampled from the waters near Tacoma, Washington, USA. The specimens were from a larger sample of Armenia collected for the Neurological Ethology course at Friday Harbor Laboratories. Healthy Armenia have longitudinal ridges along their dorsal side. When an individual becomes diseased, its skin becomes damaged so that there are irregularities in these stripes. Once this damaged is observed, their condition quickly worsens, and the individual perishes. Initially, when the Armenia were housed in a single sea table, the disease spread quickly among specimens. The infected individual have since been quarantined, and the spread has slowed, though it seems it has not yet stopped. We began our observations by making streaks on plates. The class divided into five groups, each sampling from a different specimen. Specimens were classified by two identifications: first, whether the specimen was taken from the diseased or healthy tank and whether the specimen had a lesion. My group (me, Carrie Keogh, and Tiffany Yap) investigated a diseased individual from the diseased tank. Each of us made four streaks from the specimen. The first two streaks were from a point on the leading edge of the lesion, one on an agar plate and the other on a TCBS plate. The second two streaks were from healthy skin on the dorsal side of the specimen, one on an agar plate and the other on a TCBS plate. We attempted to isolate colonies by streaking in manner such that four quadrats were streaked with quadrats two through four coming from a sample of the previous quadrat. Tools were sterilized between quadrats and plates. These plates were then incubated. More detailed instructions can be found at http://bio533.wikispaces.com/Lab_Littorina.
We also prepared scrapings from both healthy tissue and the lesion. This was done using two blades. One blade was used to hold the specimen in place, and then the second blade was slid forward towards the second blade, collecting a sample of the out cells and the mucus. These samples were then compared under a compound scope. Nothing unusual was noted in the healthy tissue scrape, but a ciliate in the shape of an ellipsoid was found in the lesion scraping at x400. A diagram was included in my lab notes. We later gram stained slides of these scrapings, which I believed were prepared by Alanna Martin. The procedures for gram staining can be found at http://bio533.wikispaces.com/Lab_Littorina. The slides were predominantly pink in color.
We also extracted DNA from the Littorina samples that we collected yesterday in order to perform PCR. The protocol can be downloaded at http://cl.ly/40f0fb74b29f4989bc49 (pg. 15). I extracted KJS8, which was tissue from the infected L. sitkana that I dissected on July 20, 2010. An error occurred in the process, likely because, as Lisa Fong noted, I used an improper P200 pipetter tip. I think this meant that I failed to pipette 200 microliters of supernatant at step 8. The result is that I extracted less DNA than intended. However, there was still the four microliters necessary to perform PCR. Information on how to generate the master mix can be found at http://aquacul4.fish.washington.edu/Protocols:Information%20Sheets/Commercial%20Protocols:Manuals/Promega_2x%20GoTaq%20MM.pdf.
July 21, 2010
Viewed the agar and TCBS plates that were prepared on July 20, 2010. Of the four plates that I prepared, two had grown colonies. The agar plate for the healthy tissue sample had a long continuous colony where ever I had swathed on the plate, thus appearing in all four quadrants. The colony had a smooth, white appearance. The agar plate for the lesion sample contained four colonies in the first quadrant. Three colonies were circular, and the fourth was shaped like a teardrop. All of them were smooth and white, like the colony found in the healthy tissue. These colonies were, however, considerably smaller in size. No colonies formed on any of the TCBS plates. However, I did examine TCBS plate prepared by Alanna Martin that contained colonies with a yellow appearance, implying that the bacteria in these colonies were able to ferment sucrose. I believe the presence of these colonies implies that her Armenia had Vibrio on it.
We also made observations of a diseased Tritonia diomedea collected for the neuroethology course at Friday Harbor Laboratories. I believe the infection has been diagnosed as a thraustochytrid. I believe the infection is characterized by pale lesions on the dorsal side of Tritonia.
We also went to Picnic Cove on Shaw Island to collect specimens of Zostera that were infected with Labyrinthula. Infected tissue has a pale coloration but can easily be confused with other forms of tissue damage (Wyllie-Echeverria, personal communication). Picnic Cove is a seagrass bed with a soft substrate bottom. An attempt was made to culture the Labyrintula , but I was not involved in that project. However, I believe the procedure was to cut the infected tissue into small pieces and places these pieces into magic medium. Cultures were also made of healthy tissue and empty medium to use for comparison. We collected samples of crustaceans (mostly, if not exclusively, Cancer magister) and cockles from the site in order to examine whether they are associated with the area's Labyrinthula infections. One C. magister did have a dark lesion on its ventral side. Carrie Keogh made a scraping of the lesion, but I do not think she had the time to interpret her scraping. A few of us bled both a healthy C. magister and a dead C. magister to compare the hemacytes, as well. The protocol for bleeding is thus:
1) Hold an uncapped syringe in one hand with your thumb just under the plunger. Be sure that the palm of the hand does not cover the bottom of the syringe and that the plunger can be moved upward with the thumb.
2) Insert the syringe into the crab's sinus (the "joint" between segments of the leg). Be sure not to stick the needle to far down or the tip will travel completely through the sinus. Once the tip is within the sinus, push the plunger upward to suck up a sample of hemolyph. Trial and error may be necessary to find an appropriate position. We know when an appropriate position has been reached with hemolyph begins to enter the syringe.
3) Once you have a sample of hemolymph, remove the sip of the syring and gently deposit hemolymph onto the slide. Cover the sample with a cover slip. These slide are now ready for inspection.
Based on bleedings from Dr. Friedman and perhaps Carrie Keogh,hemacytes sort of have an irregular shape and are relatively large. Hemacytes were plentiful in the sample from the healthy crab. However, the dead crab had few hemocytes but plentiful protozoa. Later in the evening, me, Carrie Keogh, Lisa Fung, and Lisa Crosson dissected the dead crab to check for metazoan parasites. The dissection was conducted by removing the top of the carapace using diseection scissors. In the course of these dissection, we also made presses of the gonads, caceum, neurological system, and gills. No metazoan parasites were observed, but many protozoans were present in all of the tissue types sampled.
In addition, we also extracted RNA from the snail samples prepared on July 21, 2010. I once again processed tissue from KJS8. A protocol for extracting RNA can be found at http://bio533.wikispaces.com/Lab_Littorina. To extract the RNA, I followed this protocol with only minor alterations except for the following amendments:
-At step 27, I only added 200 ul of 0.1%DEPC-H2O.
-The spectrophotometer was broken so I was unable to perform step 30. In addition, I am concerned about potentially cross contaminating.
My solution did not form a visible pellet, so I am unsure how successful my extraction was. I am also concerned about two errors that I might have committed during extraction. First, I may have bumped the region of the tube where, if I had any RNA, the RNA would be found with the pipette tip, potentially removing the RNA. Second, I am concerned that I may have cross contaminated the 0.1%DEPC-H2O. I needed to add 200 ul of 0.1%DEPC-H2O, which, due to equipment failure on July 21, I opted to add using a P100 pipetter. Clearly, this required me to 0.1%DEPC-H2O to my tube twice. However, I failed to switch tips before adding the second 100 ul of 0.1%DEPC-H2O.
July 24, 2010
Reverse transcribed the RNA that we extracted on July 24, 2010 to cDNA. The following protocol was used:
1) Heat 17.75 ul of RNA at 70 degrees Celsius for 5 minutes. Then transfer the RNA to ice, and let it cool for 5 minutes. (I used 17.8 ul in this case)
2) Generate the following Master Mix:
5 ul 5x MMLV Buffer
1.25 ul 10 mM dNTP
0.5 ul MMLV RTase
0.5 ul Oligo dT primers
3) Add the 7.25 ul of master mix to the RNA.
4) Heat at 42 degrees Celsius for one hour, and then heat the tube at at least 95 degrees celsius for three minutes. Store in a cold place when complete.
Once we had cDNA, we ran a QPCR to quantify the [relative] amount of mRNA present for c-jun kinase, using beta actin mRNA to normalize. The master mix used to run the QPCR was mixed by each student individually. Each master mix was composed of the following elements, multiplied by the number of desired reactions, in this case five reactions each for beta actin and c-junc:
-12.5 ul 2x Sybr MM (the dye)
-1.5 ul BSA
-0.5 ul forward primer
-0.5 ul backward primer
-8 ul sterile water
We then placed 23 ul of each master mix into four separate wells. To two of each four, we added 2 ul of cDNA while in the other two we added 2 ul of NanoPure seawater as a negative control. We then placed the cells into the appropriate real-time PCR machinery, and let it run. This generated both fluorescence versus cycles ran curves and a melting curve, allowing us to calculate the relative amount of mRNA for c-junc present within our samples (when standardized using the amount of mRNA present for beta actin) using the former and to determine if samples were contaminated with the latter.
Dr. Friedman also took some samples from one of the Tritonia with a thraustochytrid infection. It was a specimen from the neuroethology couse, and so the specimen was missing its brain, head, and rhinophores. While the specimen was being euthanized, it also tensed up. As a result, the exact location of the lesions could not be directly observed, so Dr. Friedman samples tissue based on a photo Lisa Fung took of the specimen yesterday. Samples were essentially chunks of flesh removed from the dorsal side of the Tritonia. In addition, Carrie Keogh and myself prepared presses of tissue from the Tritonia. We observed some tubular structures in the sample that a whitish color and a net-like appearance in the sample; we also found large circular cells that resembled photographs of thraustochytrid that Dr, Harvell showed us. However, without a sample of tissue known to be healthy, we could not determine if these observations were out of the ordinary.
July 25, 2010
Analyzed the results of the QPCR conducted yesterday. I had eight wells in total: two with actin primers and cDNA, two with actin primers and NanoPure water, two with c-junc kinase primers and cDNA, and two with c-junc kinase primers and NanoPure water. There was contamination in the actin controls, but not the c-junc kinase controls. Further, based on comparisons between other students' melting curves and my melting curves, there is likely to have been contamination in my cDNA samples as well. However, the following is my results:
| Well |
Ct |
Arbitrary Expression Value |
|
| Actin Primer/cDNA |
38.97 |
0.49682 |
|
| Actin Primer/cDNA |
35.57 |
5.2519 |
|
| Actin Primer/Control |
37.92 |
1.0292 |
|
| Actin Primer/Control |
39.09 |
0.45719 |
|
| C-Junc Primer/cDNA |
34.86 |
8.5935 |
|
| C-Junc Primer/cDNA |
ND |
ND |
|
| C-Junc Primer/Cotrol |
ND |
ND |
|
| C-Junc Primer/Control |
ND |
ND |
July 26, 2010
Prepared slides in order to use in situ hybridization to visualize WS-RLP. The protocol used is from Antonio (2001) and can be found at the following web address: http://bio533.wikispaces.com/Lab_Withering. Today we completed the following steps: tissue deparaffinization, permeabilization of tissue, prehybridization, and hybridization.
July 27, 2010
Continued preparing slides for in situ hybridization to visualize WS-RLP. The protocol followed is once again found at http://bio533.wikispaces.com/Lab_Withering. Today we completed the following sections: all the steps in the stringency wash section and the first six steps in the detection section. We left the slides overnight to make sure that detection occurred properly.
July 28, 2010
Finished preparing the slides for in situ hybridization to visualize WS-RLP. The protocol is once again found at http://bio533.wikispaces.com/Lab_Withering. Today we completed the final steps of detection (steps 7-10). According to Dr. Friedman, no group in the lab successfully stained any strain of Rickettsia, although according to Lisa, some slides probably were stained but were stained very lightly. Had the ISH been successful, the Rickettsia cells would have been dyed anywhere from dark violet to black (Friedman, personal communication).
In addition to the ISH, we also viewed H&E stains of tissues that were close to the tissues with which we attempted ISH. There are three strains of Rickettsia that be identified using H&E staining: classic, new, and stippled. Inclusions of any of these strains are ovoid masses that which are composed of multiple cells. The classic strain can be differentiated by its lighter color and smaller inclusion size. The new strain has darker inclusions and larger inclusions. The stippled strain is unique from other strains in that its inclusions have darker specks within them.
In addition we also ran SDS-PAGE for the proteins found in both heat stressed Anthopleura and Anthopleura kept at a normal temperatures. The protocol for protein extraction can be found at http://bio533.wikispaces.com/Lab_Proteomic in the Protein Extraction section. We only cut our specimens longitudinally so that every sample contained the same types of tissues. At step 7, we stored the samples in the lab refrigerator rather than storing on ice. Once the proteins were extracted, we loaded the proteins into a polyacrylamide gel. The protocol for loading the gel can be found at http://bio533.wikispaces.com/Lab_Proteomic in the Protein Gel Protocol: Extraction section, but we used 50 ul of all reagents at step 3. Each specimen had its proteins loaded into three wells. One well received 30 ul of product, the second well received 50 ul, and the third well received 10 ul. The protocol for loading and running the gel are found at http://bio533.wikispaces.com/Lab_Proteomic in the Protein Gel Protocol section. We also changed the acetic acid once during the overnight incubation period.
July 29, 2010
Inspected the gels from the SDS-PAGE ran on July 28, 2010. The gels turned out well; all the wells yielded well-stained, visible bands. Based on the quality of these bands, it was decided to use 30 ul for our Western blots. In order to perform the Western blot, we needed to generate another SDS-PAGE gel. Using the same protein extracts that we prepared on July 28, 2010, we re-did the gel following the protocol found at http://bio533.wikispaces.com/Lab_Proteomic under the Protein Gel Protocol section. Each specimen was placed into two wells, one receiving 30 ul of protein extract and the other receiving 20 ul. Once the gel was completed, we followed the protocol found at http://bio533.wikispaces.com/Lab_Proteomic under the heading Transfer Proteins to the membrane. Then we performed the Western blot. The protocol for performing the Western blot can be found at http://aquacul4.fish.washington.edu/Protocols:Information%20Sheets/Commercial%20Protocols:Manuals/Invitrogen%20-%20WesternBreeze%20Chromo%20Manual.pdf in the section WesternBreeze (trademark) Chromogenic Immunodetection Protocol: Small Gels (page 7). The primary antibody solution in this case was for heat shock proteins. We did not detect any heat shock proteins; no bands appeared.
We also dissected Ostrea edulis in an attempt to find hemocytes infected with Bonamia. To open an oyster, I tried a few techniques; I will discuss the one that worked the best. I used a serrated butter knife to whittle down the ligament in the hinge of the oyster. Then I stuck the knife into the hinge and wiggled it until the blade reached inside the oyster. Then I slid the knife around the edge of the oyster until the abductor muscles were severed, and I could pop the valves open. During this process, I was careful to keep the blade away from the soft parts so that I would not damage any tissue. Once I had opened the valves, I used a syringe to extract hemolymph from the pericardial space (located directly ventrally from the abductor muscles. I then removed the needle and syringed the hemolymph sample onto two slides, one in which I spread the hemolymph across the slide and the other in which I left the hemolymph in droplets. Emma Timmins-Schiffman helped me a great deal with these steps, as I may not have drawn from the pericardial space originally. I also removed the heart, blotted it against a paper towel and then blotted against a slide. All three slides were labeled and soaked in ethanol for one minute, and then transferred to a dye solution for a few minutes. Once they were done in the dye, the slides were removed and allowed to dry. When the slides dried, I examined them under a compound scope. In general, my slides had very little of interest on them. However, the heart blot may have contained some hemocytes. The appeared as purple circle (what I believe was the nucleus) within a larger membrane (what I believe is the cell membrane). These cells at least looked like the confirmed hemacytes that Emma Timmins-Schiffman prepared from her oyster. I did not see any Bonamia, but I did view a slide prepared by Alanna Martin that may have contained Bonamia. The affected tissues contained cells with a colored patch (assumedly the nucleus) pressed against the cell membrane. Since I did not see any Bonamia, I cleaned up after I had inspected my slides to my satisfaction, but, had I wanted further magnification on a slide, I could have waited until the slide was completely dry, then coated the material in oil, and viewed the material at 1000x using the 100x objective lens.
July 30, 2010
On July 29, 2010, the Western plot failed to reveal any bands. Dr. Roberts was concerned that this was because the protein did not transfer from the gel to the membrane, so he ran another Western plot. However, this time the blot included not only Anthopleura samples but also tissue samples from other organisms in Lab 5. The resulting blot did not reveal any binding between the antibodies with dye and HSP in Anthopleura tissue, but it did show binding with proteins from other organisms (I believe oysters, specifically). This led some students to then run more elaborate Western blots, but I do not know what they did differently in this case. Perhaps view Alanna Martin or Maire La Riviere's journls for more details.
I went to Argyle Lagoon, San Juan Island, WA, USA to collect Protothaca staminea. Previously, Huntley (2007) described P. staminea as a host for a trematode and that these trematodes cause pits in the shell of P. staminea. This observation was fundamental to a previous project that I worked on at Friday Harbor Labs. I hope to both confirm that this species is associated with pits and to identify what species of trematode infects P. staminea. I collected 28 Protothaca staminea like bivalves and housed them in a seawater tank in the lab. I dissected five of my specimens on this same day. I dissected five individuals. Of these, one individual was not a Protothaca, three had neither pits or signs of the parasite in the soft tissue, and one individual that may have had a pit, but I did not find any individuals which I could confirm were infected.
In addition, Carrie Keogh and I prepared and ran a PCR on the Littorina sitkana G-protein coupled receptor gene as a part of our group project. We wrote a more detailed account of this activity at http://bio533.wikispaces.com/Trematode+Group+Project. The hope is that these PCR products will help us to understand if the G-protein coupled receptor gene is methylated in L. sitkana.
The class also began work on the class project. I was not very active in this process as I was working on the PCR, so I am not certain exactly what was done. However, based on what I know has been done, I am guessing that Friday was largely devoted to setting up the oyster larvae in the static system.
July 31, 2010
Created an agarose gel to look at the results of the PCR that was ran on July 30, 2010. More details about this process can be found at http://bio533.wikispaces.com/Trematode+Group+Project. The first gel gave somewhat ambiguous results, so we made and ran a second gel as well. No conclusions could be reached based on the second gel either. There may have been bands. We could not tell due to the brightness of the primer dimers.
We also sampled Littorina sitkana from Cattle Point. We aimed to collect 60 large adults and 15 smaller adults from this site. The site had a great deal more birds than before, and there was an abundance of guano.
We also continued work on the group project. My job was to go to the stock room and get a squirt bottle. I am not really sure what happened while I was gone. I think the class changed the water in the static system. When I got back with the squirt bottle, they said they did not need me anymore, so I started work on the gel. I believe that someone must have began a Vibrio culture because the Vibrio needs to incubate overnight before it can be used for inoculations.
Aug. 1, 2010
Inoculated the larvae in the group project with varying concentrations of Vibrio. Two containers of larvae received only marine broth without any Vibrio in it. The first two containers received 150 mL of Vibrio stock, the next two received 1.5 mL (I believe), and the last received 15 ul. Carrie Keogh came back in the evening to photograph a sample of the larvae in an attempt to get survivorship estimates, but the results were somewhat ambiguous. Also on this day, Morgan Mouchka and Kathy Morrow cultured Vibrio on plates to determine the concentration of Vibrio in the stock. I labeled the plates and sealed them in parafilm.
In addition, my group and I dissected fifteen large adults from our Cattle Point sample collected on July 31, 2010. More details about these procedures can be found at http://bio533.wikispaces.com/Trematode+Group+Project.
Aug. 2, 2010
Continued working on the group project. My role was once again to go to the stock room, this time to buy gloves and transfer pipettes. I once again am unclear on what happened while I was away, but I believe some group members fed the oyster larvae and Morgan and (I think) Kathy counted the number of Vibrio colonies that were on the plates that they prepared yesterday. Later in the day, Emma Timmins-Schiffman and Alanna Martin determined surivorship of the larvae. Many more survived than we expected. We also had a class meeting about the class project.
We also ran serum agglutination tests onVibrio cultures following the protocol found at http://bio533.wikispaces.com/Lab_OA under the heading Serum Agglutination Test. The Vibrio positive sample formed closely packed, snow flake-like patterns. The control sample just had randomly assorted dots.
Dr. Roberts looked at our second gel from July 31, 2010, and noted that the primer dimer band was far brighter than he would have expected. Through this process, we determined that a mistake was made in the master mix; the restriction enzymes needed to be diluted before being added to the master mix so our master mix had an excess of primers. We therefore had to redo the PCR to test for methylation. See http://bio533.wikispaces.com/Trematode+Group+Project for more details.
We also added DNase to the RNA samples from Cattle Point littorines. See http://bio533.wikispaces.com/Trematode+Group+Project for more details.
Aug. 3, 2010
Ran protease assays for Vibrio spp. To do so, we followed the protocol found at http://bio533.wikispaces.com/Lab_OA under the heading Test for protease. Our original sample was from a culture of Vibrio in marine broth. The end product was indeed a bright orange liquid that differed in color from the original culture. Horray!
We also began a differential display comparing sea fans infected with various pathogens to uninfected sea fans. The cDNA libraries were already constructed for us by a member of Harvell lab, so we began by PCRing using arbitrary primers. The class was divided into two groups, one performing the PCR reactions for the infected specimens and the other performing PCRs for the uninfected specimens. Each group had five reactions, each receiving 19 ul of master mix. The master mix contained:
10 ul 5 mM arbitrary primer (#19, in my case)
5 ul 10 mM dt-ACP (adaptor primer)
30 ul H2O
50 ul See Amp 2x MX (which contained all the other agents necessary for the PCR process)
To each sample, we also added 1 ul of cDNA. Then placed the samples into the PCR machine to processed tomorrow.
For the trematode group project, we made two agarose gels that we attempted to refrigerate for later. However, we accidentally put them in the freezer instead. This ruined the gels. So we made another gel to analyze whether there was likely methylation at the G protein-associated receptor gene was methylated. Unfortunately, some of the wells in the methylation sample had dessicated overnight, so we could not load all the samples. We filled the remaining spaces in the gel with samples from the mRNA cleaned of DNA to assess whether any genomic DNA was still present. The results from the methylation samples were somewhat ambiguous, and the only sample that appeared to have contamination was a negative control. See http://bio533.wikispaces.com/Trematode+Group+Project for more details.
Aug. 4, 2010
Finished the differential display that we began on Aug. 3, 2010. I know that this entailed running a gel of the products and potentially running RT-PCR. Here are the results:
For the trematode group project, we ran a QPCR using actin primers (as a standard) and G protein-mediated receptor primers. See http://bio533.wikispaces.com/Trematode+Group+Project for more details.