PCR and Gel Electrophoresis

I used to teach a genetics lab and thought I would share what I taught my students. Before running polymerase chain reactions (or PCR), to make copies of your sample DNA, it is important to run a gel with your DNA extraction to see if the extraction worked. To run a gel, the DNA sample is loaded into wells on one side of a gel. An electrical current is used to separate out the DNA to analyze it. If your DNA extraction did not work it is a waste of money to run PCR on the sample. This gel is run in buffer TBE. TBE contains tris which raises the pH to 9, a boric acid which lowers the pH to optimizes it for the target enzymes, and EDTA which chelates metal ions and those stops DNeasy activity, which is the enzyme used for DNA extraction. The molecules in the gel separate based on charge, size, and shape. Personally, I like to use 1% agarose gel with TBE at 120 volts for 30 minutes.

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But what is agarose? It’s a seaweed polysaccharide that allows the gel to thicken. The more agarose you use the thicker the gel and the longer it will take your DNA sample to run through the gel. If you need to separate out really small fragments you would need a really thick gel but since we are looking to see how well the DNA extraction worked we can use a thin gel. You should also stain the DNA sample before putting the sample in the gel. If you don’t stain the sample you wouldn’t be able to see where your sample goes in the gel and run the risk if running your sample off of the gel. Gels also often have glyercide in them that binds to the DNA and makes it heavier so when you load your DNA into the well on the gel it is less likely that the DNA floats away. It another well a ladder is also typically used. In this case, we used a 1000 kb base pair ladder. A ladder is DNA fragments that are cut at known lengths. This allows it to act as a ruler for your other samples.

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Queen parrotfish (Scarus vetula)

The male queen parrotfish (Scarus vetula) is one of the most striking fish in the Carribean and happens to be my 2nd favorite fish. Females are a drab blue brown color while males are green-blue. This is an example of sexual dimorphism. Both sexes have plate-like beaks which give them the name parrotfish. They use these beaks to break off coral covered in algae. they then chew that mass and excrete the matter that isn’t algae. This process creates sand from the coral they ingest and is actually a major way in which sand is produced. This grazing also opens up space for coral to grow back in areas overtaken by algae. In areas without parrotfish reefs have been shown to shrink because coral can’t out-compete algae. They also feed on sponges and other creatures that may be attached to the reef. The fish is also known by the names blownose, blue chub, blueman, blue parrotfish, Joblin crow parrot, moontail, Okra peji, and slimy head. They are native to reefs in the Carribean, and thus also restricted to shallow water. 1622-parrotfish_0

The fish breed throughout the year but typically restrict breeding to mornings. The fish exercise harem polygyny, which means that one male mates with a harem of females. They school in groups with one male and 3-4 females. However, the fish are also protogynous hermaphrodite which means that they can change their sex from female to male. In fact, all queen parrotfish are born as females. As they mature the largest in the school becomes male. The male that mates with the females is called a supermale and is identifiable by his bright coloration. The male chases the females and they swim in tighter and tighter circles until they release their gametes and the eggs are fertilized through external fertilization.

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A female queen parrotfish (Scarus vetula)

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The genetics of viruses

Viruses are infectious particles made of nucleic acid encased in a protective protein coat and, sometimes, a membranous envelope. The genome of viruses may consist of double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA, depending on the kind of virus. The viral genome is usually organized as a single linear or circular molecule of nucleic acid. The genome is encased in a protein shell called a capsid which is derived from the host cell. The most complex capsids are found in viruses that infect bacteria, called bacteriophages or phages. A virus has a genome but can reproduce only within a host cell. Dr. Beijerinck used the sap from one generation of infected plants to infect the second generation of plants that could, in turn, infect future generations. Dr. Beijerinck determined that the pathogen could reproduce only in the host, could not be cultivated on nutrient media, and was not killed by alcohol which generally kills bacteria.

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An isolated virus is unable to reproduce—or do anything else, except infect an appropriate host. This is because viruses lack the enzymes for metabolism and the ribosomes for protein synthesis. Each type of virus can infect and parasitize only a limited range of host cells, called its host range. Viruses identify host cells by a “lock and key” fit between proteins on the outside of the virus and specific receptor molecules on the host’s surface. Most viruses of eukaryotes attack specific tissues. Most DNA viruses use the DNA polymerases of the host cell to synthesize new genomes along the templates provided by the viral DNA.

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The Nutritional Ecology of Bird Migration

 

I attended a lecture by Dr. Susan Smith on her research related to migrating birds and berry nutrition. Migratory birds spend up to four months a year in the process of migration. These migrations are to wintering grounds and breeding grounds. During these trips, physiological demands are much greater. These trips require very large energy reserves. 79 percent of these reserves come from fats.

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These stores of energy are depleted and then restored at migratory stopovers. At these sites, refueling occurs. This must be rapidly done due to time constraints (must get to wintering ground before winter). Bird’s diets change during migration to be able to rapidly refuel. Birds eat large amounts of fruit. The benefits of eating fruit in place of their normal diet are fruit is high in fat and fruit is easier to hunt. There are limits birds have to eating fruit. One type of limitations are digestive. Bird’s digestive systems can only handle a certain amount of seed load. Also, some fruits contain a certain amount of toxins and the system can handle only so much. Other limitations are nutritional.

Fruit varies in energy and protein content. Some contain up to 40 percent fat, most of these include native species. As fat content increases, energy density also increases. A hermit thrush (Catharus guttatus) (that on average weighs 31.2 grams) would have to eat 18.8 grams of bayberry (Myrica sp.) (high in fat) or 90.7 grams of pokeweed (Phytolacca decandra) to fill the daily energy needs. Eating this much pokeweed is clearly not possible.

 

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A hermit thrush (Catharus guttatus)

 

One of Dr. Smith’s studies tried to see if birds prefer certain fruits during autumn migration time. Nets were placed around berry bushes and the amount that natural falls off was measured. The nets were removed and the amount of berries eaten during different times of the year was measured. The amount of arrowwood (Viburnum dentatum) (high in fat) eaten increased during migration, were as the amount of chokeberry (Aronia sp.) (low in fat) eaten stayed the same.

Another study was done by Dr. Smith using the plasma metabolic profile (blood samples). These samples provide information on metabolic fuel use and energetic condition. Lipid metabolites indicate how mass has changed over the last several hours (showing refueling). Other metabolites provide more information. Plasma was also sampled at two different stopover sites to compare them.

These sits were the Braddock Bay Bird Observatory and Rochester Institute of Technology Bird Observatory. At Bradock Bay Bird Observatory there was 200% more ripe fruit and this was 83% native berries (tend to be higher energy). At Rochester Institute of Technology, there were only 10% native berries. Birds were sampled the same day and same time at these locations. It was found that at Braddock Bay Bird Observatory those birds had higher triglyceride levels. This proves that fruit is helpful in birds refueling.

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Dr. Smith hopes to continue in researching the effects of fruit on birds. One study she wants to do is compare seasonal and site differences in fruits nutritional content. She also wants to look at if the bushes that provide fruit provide a year round value (such as a place to nest in or find insects). She wants to develop fruit fingers prints (light that comes off fruit when you shine a fluorescent light on it). This can be used to quickly tell if a fruit has high or low nutrients. Lastly, Dr. Smith wants to see if invasive species are so integrated into the system that removing then would cause harm to bird by removing a food source.

Ecosystem roles in tropical rainforests

Primary producers in all of these ecosystems vary. In rainforests, the primary producers include trees, shrubs, and epiphytes. An epiphyte is a plant that grows on another plant but it not directly parasitic. In mangroves, they are epiphyte and mangroves. The term mangrove describes how the trees live, it is not a taxonomic term. It simply describes a tree that lives in salt water. In a reef, the primary producers are corals, seagrass, and macroalgae (seaweed). Interestingly corals are also predators of the reef. The algae they are in a symbiotic relationship (zooxanthellae) with undergoes photosynthesis during the day. At night they hunt small organisms in the water and spear them with tiny barbs.

 

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A view of Guatemala primary producers. Captured by Lauren Schramm

 

Herbivores (or primary consumers) in the mangroves, rainforest, and reefs all vary. In rainforests, these include birds, monkeys, agouti, tapir, butterflies, and sloths. In the mangroves there are fewer herbivores, these include mollusks and crabs. In the reef system, most organisms are herbivores. These include coral, smaller fish, sponges, plankton, and mollusks.

 

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An Agouti (Dasyprocta punctata )

 

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Wetland delineation

The purpose of doing a wetland delineation is to establish the wetland boundary. This can be done either using the soil or the vegetation in that area. The US Army Corps of Engineers manual on Wetlands Delineation is considered the standard although there are different supplemental manuals depending the region that you are in. It is mandatory to use the manual to identify and delineate wetlands that could be subject to regulation under Section 404 of the Clean water act. While mudflats are protected under section 404, the manual only deals with vegetated sites, aka wetlands. Both the list of plants and soils listed in the manual are considered obsolete. A newer list of plants is available here and a newer list of soils is available here.

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In order to defer something as a wetland it has to meet three key criteria; saturated soils from a permanent or periodic inundation of groundwater, vegetation adapted for life in saturated soils, and the presence of “normal circumstances”. More specifically soil must be classified as hydric and the mean water depth is less than 6.6 feet deep. Wetlands may be surrounded by either deepwater aquatic habitats or nonwetlands. See the Army Corp manual on pages 10 and 11 for more specific definitions of these habitat types.

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Order Lepidoptera

The order Lepidoptera contains butterflies and moths. There are over 180,000 species in the order which is divided into 126 families and 46 superfamilies. They account for 10 percent of known species worldwide. The order is defined by the scales that cover them, wings, and a proboscis. They also undergo a complete metamorphosis between the larvae and adult stage. Some scientists have even theorized that caterpillars and adults are different species and metamorphosis changes the genes that are active. This is known as the death and resurrection theory. Butterflies and moths are important pollinators and members of the food chain. Because there are so many families in this group I have chosen to just focus on 5 families for now.

  • Saturniidae
    • this is a moth family with over 2,300 described species in it
    • adults have large bodies and wings
      • the bodies have hair like scales on them
      • wings often contain eyespots
      • front and back wings overlap
      • wingspans are typically 1-6 inches but the atlas moth (Attacus atlas) has a wingspan of 12 inches!
    • notable members include the giant silk moths, emperor moths, and royal moths
    • this family contains some agricultural pests as well as the moths that spin silk
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An atlas moth (Attacus atlas)
  • Papilionidae, or the swallowtail butterflies
    • this family has over 550 species
    • while most species are tropical these butterflies can be found on every continent but Antartica
    • the family included the largest butterflies in the world with the largest being Queen Alexandra’s birdwing (Ornithoptera alexandrae) which can have a wingspan of 9.8 inches
    • the larvae in this family have a defend organ called an osmeterium

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