S-STEM/TRAIN Scholar Jasmine Patricio

Happy Thanksgiving to everyone and happy holidays to everyone as well. Today we decided do a potluck with the interns that are working with Josh, Matt & Cory. Our research Project Draft was due this week, although I was done with everything the only thing I have left to do a conclusion to everything. Though I am a little confused how to do the conclusion to Evey thing. I have decided to redo my extraction for SM, since the outcome didn't come out to make scene.

Nanodrop (Micrococcus Luteus) Black Cap #1,2 = DI-H20 + M.L.+ Quick Extract+ E.W.L Clear Cap #1,2 = Quick Extract + E.W.L Today, I performed a nanodrop on my new bacteria Micrococcus Luteus. My focus was on the nucleic acid. Nucleic acids are the main information-carrying molecules of the cell , and, by directing the process of protein synthesis, they determine the inherited characteristics of every living thing (n.p). The goal was to get around 200-280. Seeing that the two BlackCap are the closest to the numbers indicated means that there was more possible DNA in the Bacteria than ClearCap #1,2. A 260/280 ratio of ~1.8 is generally accepted as “pure” for DNA; A ratio of ~2.0 is generally accepted as “pure” for RNA. Reference: "nucleic acid". Encyclopædia Britannica. Encyclopædia Britannica Online. Encyclopædia Britannica Inc., 2016. Web. 15 Nov. 2016 <https://www.britannica.com/science/nucleic-acid>. Desjardins, Philippe, and Deborah Co

Jasmine Patricio Palacios Instructors: Joshua James & Matt Haberkorn October- December, 2016 Develop a protocol that will involve the extraction of a Chicken-Egg White to create Lysozyme. The synthesis of the Egg White Extraction and DNA Extraction from Serratia Marcescens and Micrococcus Luteus was performed to verify if it is possible to create Lysozyme from a chicken egg white, and see how much of the DNA was purified. Summary: The protocol for purifying lysozyme was highly improved with more detailed measurements added to it to be understood by others. The bacteria S.M. and M.L. had possible DNA Extracted from them, with the DNA Extraction Kit. Both bacteria then were measured on how purify they are buy the NanoDrop. Both bacteria had the same amount of sampled measured which included 2 with the cell pellet removed and 2 with it not removed. Summary of Relevant data and results: The removal of the cell pellet compared to the sample that did not have the removal

Nano Drop Graph Open the app Click “Nucleic Acid” Open arm Add 2 ul of TE Buffer Close arm Click “Blank” Open arm Add 2 ul of bacteria on top of the TE Buffer Close arm Name it Click measure Clean off Repeat for as many samples you have A260/280 ratio The A260/280 ratio is generally used to determine protein contamination of a nucleic acid sample. The aromatic proteins have a strong UV absorbance at 280 nm. For pure RNA and DNA, A260/280 ratios should be somewhere around 2.1 and 1.8, respectively. A lower ratio indicates the sample is protein contaminated. The presence of protein contamination may have an effect on downstream applications that use the nucleic acid samples. Black cap 1 & 2 = No removal of the pellet, so that mean in the two little ones they each contained DI H2O + S.M. + Quick Extract + EWL (Egg White Lysozyme) No cap 1 & 2 = Removal of pellet, the two little tubes contain Quick Extract + EWL (Egg White- Lyso

Partial purification of lysozyme 1.        For the partial purification of lysozyme, egg whites, carefully separated from the egg yolks, were diluted 3- or 3.3-fold with 0.05 M NaCl solution. 2.       To precipitate the egg  white proteins other than lysozyme, the pH of this mixture was set to 4.0 by carefully adding several drops of 1 N acetic acid and it was diluted with an equal volume of 40% (v/v) ethanol. After 30 minute incubation at room temperature in the presence of ethanol, the mixture is centrifuged at 15,000 x g for 15 min at 4 °C; then the precipitates were discarded. 3.       The supernatants were analyzed for their lysozyme activity and protein content to determine if lysozyme extraction was successful. 4.       A DNA extraction is performed using a kit the epicenter QuickExtract Bacterial DNA Extraction Kit (QEB0905T) and E.Z.N.A. Bacterial DNA Kit from Omega bio-tek (D3350-00) as controls 5.       A DNA extraction is performed using both kits but rep

My next step after purifying the lysozyme is to run it in the PCR. The only problem that I had is that we don’t have Master mix. I can’t run the experiment without Master mix without it because that is needed for me to run it in the PCR machine. PCR stands for Polymerase Chain Reaction, which is often used in biological and chemical labs. A thermal cycler, or PCR machine, has the ability to produce DNA copies of a specific segment that can range from thousands to millions in numbers. The basic function of this machine is copying the sections of DNA and this is performed through a heating cycle. This is performed when the temperature rises to 95 degree Celsius which in turn melts the DNA strands. This melting of DNA strands causes the backbones of sugar phosphate to split apart. Then as the temperature lowers, the primers bind them 3 inch end of each sequence of target. Primers are able to perform this task as the DNA polymerase taq and free nucleotides aid it in the process. This proc

Lysozyme is an enzyme that catalyzes the destruction of the cell walls of certain bacteria, occurring notably in tears and egg white. Lysozyme breaks down the bacterial cell walls and is used to extract recombinant proteins or DNA from bacterial cells. Also used as an antiseptic to prevent caries and treat infant formulas. Like mentioned before it is found in tears, saliva and mucous. It wasn't until 1922 that a Doctor named Alexander Fleming (1881-1995), observed the antibacterial action of Lysozyme when he treated bacterial cultures with nasal mucus from  patient suffering from a head cold.   Serratia Marcescens is an opportunistic pathogen that cause nosocomial infections. It is resistant to many antibiotics traditionally used to treat bacterial infections, such as penicillin and ampicillin [9]. This is due to all of Serratia marcescens ’ characteristics; unique membrane (LPS) as a Gram-negative bacteria, the ability to survive in aerobic and anaerobic conditions, and its m