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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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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Passenger pigeon genetics

Unlike other species, passenger pigeons (Ectopistes migratorius) faced a recent extinction. This extinction took place at the turn of the 20th century, the last individual died in captivity in 1914. Based on morphology it was assumed that they belonged in the genus Zenaida (Fulton 2012). This genus consists of mourning doves. When the mitochondrial DNA was analyzed it was found that they are more closely related to Patagioenas which are the New World pigeons. They used mitochondrial DNA because there is a high number of copies of this DNA in the cells when you compare this to the 2 copies of DNA that are in the nucleus. For this reason, it is common to use this for the analysis of ancient DNA.

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BT corn

The following is a paper that I wrote for my population genetics class. 

Corn is a large part of the diet of North Americans. Furthermore, 15 percent of the land coverage of genetically modified crops contain Bt proteins (Hoftmann 2011). Bt corn was created because there is a high demand for corn that is damage free. Corn pests include wireworms (Elateridae sp.), cutworms (Noctuidae sp.), European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), and white grubs (Phyllophaga sp.). In the past, the soil had to be treated before planting in order for the plants to be unaffected. Affected plants had stunted growth which led to a reduction in their yield. Not all pre-planting treats were effective either. (University of Kentucky) In the past, in order to deal with these pests crop rotation was also used. The areas most affected by these pests were Illinois, Indiana, Michigan, Ohio, and Iowa.

 

Bt corn first appeared in 1996 in Zurich Switzerland. Monsanto had the corn in development since 1992. The genes cry1Ab, bar, and bla were put into the corn. The cry1Ab gene comes from Bacilus thuringiensis, bar from Streptomyces hygroscopicus, and bla from Esherichia coli. Cry1Ab makes the crop resistant to lepidopteran species (butterflies and moths) by damaging midgut lining. It breaks down the gut wall which allows bacteria to flood into the body cavity. Bar makes crops resistant to herbicides by eliminating the activity of glufosinate in cells. It does this by acetylation. Bla detoxifies beta lactam antibiotics. This paper will focus on the bar insertion that causes resentence to pests, the Bt gene. Current the crop is used for food in 13 countries, feed in 10, and cultivation in 4 (International Service for the acqaution of agro-biotech applications.) Currently, Monsanto is working on increasing the resistance with corn rootworms (Diabrotica virgifera) and pink bollworms (Pectinophora gossypiella) (Monsanto 2015).corn_borer

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Avian eggshell formation

Many birds, more specifically passerines (order Passeriformes), rely on different sources of calcium for skeletal growth and store calcium in their bodies, a necessity for their ability to form strong eggs during the eggshell formation process. Near human settlements birds and in areas with high qualities soils, birds tend to have more access to the calcium that they need. Because of the importance of calcium passerines often ingest snail shells in areas in which the soil is calcium deficient. The most common source of calcium is snail shells followed by calcareous grit, bones, eggshells, and other sources.  Soil can be calcium deficient can be caused by acid precipitation which removes calcium from the soil. Most passerines are unable to ingest their much-needed amounts of calcium due to the scare amounts of nutrients in the habitats that they live in. This results in the development of thin fragile eggs that are unable to hatch thus affecting the bird population in various habitats.

 

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 Burgundy snail (Helix pomatia): a common European species

 

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The Cell Cycle

In the cell cycle, cell division functions in reproduction, growth, and repair. The two forms of cell division are mitosis and meiosis. Mitosis requires the distribution of identical DNA to two daughter cells. Meiosis yields four nonidentical daughter cells, each with half the chromosomes of the parent. The purpose of meiosis is to produce cells for reproduction, and as you want the offspring to have the same number of chromosomes as the parents’ sex cells need to have half the number of chromosomes as the parents. A cell duplicates its DNA, moves the two copies to opposite ends of the cell, and then divides into two cells.  This genetic information that makes up the cell’s genetic information is called its genome.

The genome is made up chromosomes. Every eukaryotic species has a characteristic number of chromosomes (packaged DNA) in each cell nucleus. Eukaryotic chromosomes are made of chromatin which is a complex of DNA and protein. When a cell is not dividing chromosomes are chromatin fiber in the nucleus. Each duplicated chromosome consists of two sister chromatids, which appear when cell divisions are about to take place. The chromatids are attached at what is called the centromere. After the chromatids divide mitosis is followed by division of the cytoplasm or cytokinesis.

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Coliform bacteria testing: a how to

Coliform bacteria are associated with water that is unfit to drink as they come from fecal matter. Ideally, our drinking water would have no coliform bacteria in it, otherwise, water should be boiled to drink. Being able to perform your own coliform bacteria test is ideal if you are a well owner or have a water body on your property. It is fairly easy to do but does require some startup cost. There is a growing movement of DIY Biolabs that may have equipment available for use. The method that I will be outlining is the membrane filter method (EPA method 1604). In this method, water samples are filtered through a membrane that retains bacteria and then the samples are placed on growth medium and incubated. The bacteria grow dome-shaped colonies which are then counted after 24 hours. Please note that I am not an expert, and this article should not be used entirely for testing. Please read the EPA method and if you feel you don’t fully understand the method then have your drinking water tested by a lab.

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