The document summarizes several biology lab experiments conducted by the author: 1. They performed DNA extraction from samples, PCR, and Western blot techniques. For DNA extraction, their sample did not produce the expected results. 2. They learned aseptic technique and used Gram staining to identify bacteria samples as gram positive or negative. 3. PCR was used to amplify genomic DNA between primers over multiple cycles. Controls were included. 4. Nested PCR with more specific primers was used to further amplify portions of DNA. Exonuclease treated samples before nested PCR. 5. Gel electrophoresis separated DNA fragments by size. PCR products from two plant samples were analyzed, with one showing bands.

1. Maralys Colón Betancourt<br />804-09-1419<br />RISE1<br />Paragraph #1<br />Laboratory from North Carolina<br />(February 12 +13 , 2010)<br />In this workshop we realized a wide variety of experiments like extracting DNA, performing PCR, realizing Western Blot and others. In the extraction of DNA we used commonly used materials to lyse the cell and denature the proteins. Even though the final result should have been a white stringy DNA, mine didn’t look anything like that; it broken down. Some of the reasons for this result could have been that I didn’t shake the tube very well or that I didn’t have many cells in my mouth because something that I ate might have killed them (an acid substance). The PCR was a technique we used to run the mouse DNA. In the Western Blot we pipetted the samples (a mixture of loading buffer, protein lysate and water) into the wells of the electrophoresis chamber for the detection of proteins. In the agarose gel, we prepared the samples and introduced them in the wells for the separation of the DNA fragments based on size, shape, and charge.<br />Paragraph #2 <br />Laboratory #4: Aseptic Technique and Gram Stain Method<br />(February 19, 2010)<br />The purpose of this lab was to understand and use the aseptic technique for the improvement of the final results of an experiment. By using this technique, the area of work will be clean and purified. Also, we used the Gram Stain Method for identifying which sample (A or B) was gram positive or gram negative. This is possible because this technique gives gram positive and gram negative different colors; this is why this procedure is referred to as a differential staining technique. Sample B was gram positive given that it retained the color of the crystal violet, which means that the bacteria have a cell wall composed of a thick layer of a particular substance called peptidologlycan. In the contrary, sample A was gram negative given that it lost the crystal violet color, which means that the bacteria have a cell wall composed of a thin layer of peptidologlycan. Another experiment we realized was the streaking of a plate. This is realized in a specific way so the petri dish is covered but that the areas have different concentrations of the bacteria. <br />Paragraph #3 <br />Laboratory #5: GAPDH PCR: Initial PCR<br />(February 26, 2010)<br />The purpose of this lab was to amplify the genomic DNA between the primers. This was realized by utilizing a technique called Polymerase Chain Reaction, better known as PCR. What this technique does is that it increments the genomic DNA in every cycle. That’s why the microcentifuge tubes were left in the thermal cycler for forty cycles. A thermal cycler is an apparatus used to amplify segments of DNA; it raises and lowers the temperature of the samples. This causes the denaturation, annealing and extension of the sample. There were seven microcentrifuge tubes per group. Four of those samples contained DNA (two for Ruellia and two for Begonia) while the other tubes were used as control negative and positive. For the negative control we used water and for the positive we used Arabidopsis and pGAP. <br /> <br />Paragraph #4 <br />Laboratory #5: GAPDH PCR: Nested PCR<br />(March 4, 2010)<br /> <br /> The purpose of this lab was to amplify portions of the GAPC gene from gDNA of the Begonia using as a template the PCR products of each gDNA sample amplified in the Initial PCR. The Nested PCR uses a more specific set of primers that will amplify the desired portions of GAPC. Nested PCR is used because it reduces the contamination in the products due to the amplification of unexpected primer binding sites. Before performing the nested PCR, we had to remove the primers from the products. For this we used exonuclease, an enzyme that specifically degrades single-stranded DNA. This enzyme needs to be inactivated after it degrades the single-stranded DNA to prevent further degradation of the nested primers. After the microcentifuge tubes were prepared, they were left in the thermal cycler for forty cycles. In the Initial and Nested PCR, the cycles are much alike yet in the annealing process the temperature varies depending on the PCR. In the Initial PCR, the temperature is 52 ˚C while in the Nested PCR it is 46˚C. There were seven microcentrifuge tubes per group. Four of those samples contained DNA (two for Ruellia and two for Begonia) while the other tubes were used as control negative and positive. For the negative control we used sterile water and for the positive we used Exo Arabidopsis gDNA and pGAP.<br />Paragraph #5<br />Laboratory #6: Electrophoresis, DNA Purification and Ligation<br />(March 5, 2010)<br /> <br /> The purpose of this lab was to use an agarose gel electrophoresis to separate the DNA fragments by size. This was done by loading the initial and nested PCR products into the gel with a loading dye. Also, if the samples had DNA, this process identifies it by the presence and intensity of the bands. From our two plant samples only one showed the band (plant #2); that’s the one we are going to use for the clonation of the GAPC. After this process, we proceeded with the PCR purification. This is needed given that it eliminates the excess of nucleotides, DNA polymerase, primer-dimers and unused primers. We used ion exchange and size exclusion chromatography for the purification. Ion exchange chromatography separates molecules based on charge while size exclusion separates molecules by size. After the samples have been purified, we ligated the gene into a plasmid or cloning vector. What this does is that it allows the propagation of the fragment. In the next laboratory, we will be introducing the plasmid into living bacterial cells, a process called transformation. <br /> <br />Paragraph #6<br />Laboratory #7: Nematodes<br />(March 12 - 13, 2010)<br /> <br /> The purpose of this lab was to prepare a cross plate of a nematode called C.elegans. For this, we had to learn to distinguish between the two sexes. The male and the hermaphrodite have a mouth but on the other extreme, the male has a structure called a fan (which looks like a triangle) while the hermaphrodite has a whip. That’s the main feature for distinguishing between them. C. elegans are very useful as models for the exploration of neuronal activity because they have many common synapses with mammalians and have a define nervous system containing exactly 302 neurons. In the lab we prepared the cross plate by moving a male and hermaphrodite C.elegans to another plate. This was hard given that I pressed the platinum band too hard and killed a lot of the nematodes. After I understood how it worked, I was able to transport nematodes easily from one plate to another; the hard part was to distinguish the sexes. Also, many of them were still at an early phase of development that didn’t show if they were male or hermaphrodite. <br />Paragraph #7<br />Laboratory #8: Transformation<br />(March 26, 2010)<br />The process of introducing a plasmid into living cell bacteria is called transformation. This is the process we used today to introduce the plasmid and to replicate it as the bacteria multiplied. The purpose of this lab was to transform the bacteria with the plasmid given that during ligation many different products are obtained, including the desired PCR fragment. This fragment might be ligated with itself, that’s why to separate the desired vector-PCR product we needed to transform it. First, the E.coli passed through a series of steps to become competent cells. The steps were: peletting the growing bacteria, cooling, washing, and resuspending it. The competent cells are then added to two microcentrifuge tubes containing the ligation reaction and labeled ‘pGAP TF’ and ‘TF’. After heat shock, the two solutions are pipetted from the tube and into their corresponding LB Amp IPTG agar plates. Finally, the agar plates were placed in an incubator and if the bacteria grow, we will then be able to continue our laboratory.<br />Paragraph #8 <br />Laboratory #9: Got protein?<br />(April 9, 2010)<br />The purpose of this lab was to quantify the proteins in two milk samples. For this, we had to dilute the milks using 1x PBS. Then, we had to add the Comassie G250 blue dye which binds to the side chains of specific amino acids. After adding the dye, we estimated the protein concentration by comparing the colors of the two unknown samples with the color of the standard samples (#1-#7). A spectrophotometer was employed to determine the exact concentration of proteins in the standard samples, in the control and the two unknown samples. Also, we were able to compare the amount of protein we obtained from the experimental procedure with the amount of protein in the food label of the milks. In sample A, soy milk, the experimental results showed that there were 66.5mg/ml while the food label had a value of 29.17mg/ml. In sample B, whole milk, the experimental results indicated that there were 76.5mg/ml while the food label indicated that there were(here goes the data from the food label, the other group didn’t have the value that day) mg/ml. This comparison shows a great difference given that in sample A, the amount in the food label is less than half of the experimental value, meaning that in reality when we drink milk, we are obtaining more protein per milliliter than the amount indicated. The same analysis can be made for sample B.<br /> <br />Paragraph #9<br />Laboratory #10 and 11: Protein Extraction from Muscle, Electrophoresis, Semi-Dry Transblot and Staining. Western Blot for the Detection of Myosin. <br />(July 7 +9/2010 )<br />The purpose of this lab was to identify a particular protein called Myosin. For this, we had to extract the DNA from the fish samples (Red Snapper, Salmon, Dorado and Tilapia). Then, we had to electrophorese the samples with a control called Actin Myosin and a Molecular Weight marker. When time passed, we were able to observe that the samples had proteins in them given that there were bands in the gel. Two of the membranes were stained with Comassie blue while the other two membranes were used in the next laboratory in the Western Blot. The Western Blot was the final step in which Myosin was detected by antibodies. One antibody called primary antibody detected Myosin while secondary antibody detected the antibody that detected Myosin. The gels showed degradation in the lanes that contained salmon. The results showed that Myosin was detected in gels number three but in gel number four no clear results were obtained. An explanation for this is that the gel looked dehydrated when it was used. <br />