Agare Gel Lab Report

The identification of unknown organisms carries important ramifications that can be applied to many real world scenarios. In keeping with quality assurance beverages, food, cosmetics, and other products are frequently inspected for contaminants resulting from a presence of pathogenic bacteria. In medicine, a physician’s diagnosis and consequent treatment is largely determined from samples collected from infection sites that have been analyzed using microbial tests.

Before beginning this experiment, the completion of an IRB Form and a Written Consent Form for the usage of human participants. After Bethel College agreed to approve this experiment, the testing began. The Bethel College Biology Department approved the materials needed for this experiment. These materials were easily attained from Wal-Mart located in Newton, Kansas which included: three bottles of Listerine Zero Total Care ®, two one hundred count packs of one ounce Kroger ® disposable bathroom cups, one two hundred-fifty count pack of Kroger ® plastic spoons, and one twenty-nine fluid ounce tub of Tropical Life ® Organic Extra Virgin Coconut Oil. The Bethel College Biology Department supplied a laboratory that housed the equipment needed

Research Question: How does the size of the cell affect its efficiency in exchanging substances through several ways, like diffusion?

When isolating the types of bacterium that is gram-positive, motile, halophilic, mannitol fermenters that grow in the presence and absence of oxygen you would use the mannitol salt agar (MSA), mannitol fermentation, and semisolid stab tests. To isolate a halophilic bacterium, mannitol salt agar (MSA) can be used as a test. For a mannitol salt agar (MSA) test, you would need a plate where the agar is mannitol salt and 7.5% NaCl. The 7.5% NaCl mix with mannitol shows the selective and differential part of the test because some bacterium is not able to tolerate the high level, but can be mannitol fermenters.

This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

Mac Conkey Agar is selective and differential culture medium for bacteria, which is designed to selectively isolate gram-negative and gram-positive bacteria and also differentiate groups of gram-negative bacteria based on lactose fermentation. The crystal violet and bile salts in Mac Conkey agar is used to inhibit the growth of gram-positive organisms which allows for the selection and isolation of gram-negative bacteria. However, for differential medium Mac Conkey agar will contain lactose, a fermentable carbohydrate, and the pH indicator neutral red. When, bacteria utilize lactose as carbon source the acid products will produce and change neutral red from yellow in neutral solution into pinkish red in the present of acidic environment. Based on the experiment, Mac Conkey agar is used to differentiate and distinguish between two groups of gram-negative bacteria which are Pseudomonas Putida and Escherichia Coli either it is lactose-fermenting bacteria or lactose

Indicator ingredients are dyes and stains and they are usually used in differential medias to show difference in bacteria’s. There were two indicators used in the MacConkey Agar plate, the first one was the PH indicator. It shows the difference in PH levels by changing the color of the bacteria. The second indicator that was used in the same media is the crystal violet and from the previous lab we can all agree that this dye was the primary dye we used to stain the cell wall of an gram positive bacteria. Cystal violet shows the difference in gram positive bacteira from the negative

The test depends on the digestion ofglucosetoacetylmethylcarbinol. If glucose is being broken down, it will react with alpha-naphthol (VP reagent #1) and potassium hydroxide (VP reagent #2) to form a red color. Alpha-naphthol and potassium hydroxide are chemicals that detect acetoin. The VP test detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide(KOH) to diacetyl.

Each test performed in the lab on theses unknown bacteria have a very specific significance. With each test performed correctly the lab officer is able to move closer towards a proper identification of the unknown bacteria. After performing Gram staining and deciphering if the unknown was gram positive or negative the lab officer was then able to proceed to the next step of identification. The gram positive unknown’s reaction to the catalase test informs the tester of the Genus theyre working with. This indicates which tests to perform next. The MacConkey agar is a selective medium that only allows the growth of gram positive bacteria confirming the results received from the gram staining procedure. The NaCl growth test indicates whether or not the organism is able to

To determine if our DNA sequences were properly extracted and amplified, we used gel electrophoresis. A 1% agarose gel was made using 1 gram of agarose powder per 100 ml of 1x TBE buffer. The solidified gel was placed in the electrophoresis tank containing 1x TBE buffer. Our samples were prepared by mixing gel loading solution with our PCR products (3 µL gel-loading solution, 5 µL PCR product). The samples were loaded in a selected order (table 2) and the gel was run between 30 minutes to an hour at 70-80 V (Lab 8, 2017)

In this investigation, 2-aminophenol(AP), 4-chloroBenzaldehyde(CB), the metal salts CuCl2.2H2O(99.00%), Pd(OAc)2(99.99%), and AgNO3(99.50%) and Calf thymus DNA(CT-DNA) were utilized starting chemicals and purchased from Sigma–Aldrich Chemise (Germany). Agarose was purchased from Fischer- Biotech (GE Heath care). Tracking dye (0.25 % bromophenol blue, 40 % sucrose, 0.25 % xylene cyanole and 200 mM EDTA) and Ready-load 100 bp DNA ladder were used as the native size DNA and purchased from Bio labs.

The chemical and reagents used for the extraction and quantitation of DNA were: Plant DNAzol (0.3ml/0.1g), 100% ethanol (100%: 0.225 ml/0.1 g, 75%: 0.3 ml/0.1 g), Chloroform (0.3 ml/0.1 g), Plant DNAzol-ethanol solution: Plant DNAzol, 100% ethanol (1:0.75 v/v), TE buffer (10 mM Tris, 1 mM EDTA pH 8.0), 1.2% agarose gel (Agarose, 1X TAE buffer), 6X loading buffer (glycerol, Tris/EDTA pH 8.0, ethidium bromide), .25X TAE buffer, Restriction enzymes and Restriction endonuclease buffers. All the chemicals used were quality grade. The restriction

The materials used for the study are the following: sterile swab, eppendorf tubes, 0.2 M NaOH solution, 0.04 M Tris solution, PCR sample loading dye, 1% agarose gel, DNA marker solution, PCR MasterMix, Homozygous and Heterozygous control solutions.

The fragments were amplified using Phusion High-Fidelity DNA polymerase with standard reaction conditions and either genomic or plasmid templates. All fragments shown on the gel are of the expected size indicating the correct fragment has been amplified. The expected sizes are indicated on the gel with mCherry 737 bp, TtrpC 589 bp, noxA 1976 bp, mssD 3097 bp and PgpdA 2338 bp. After gel extraction and purification mCherry was shown to be of very low concentration 13.5 ng/L by the nanodrop. This was not sufficient for successful Gibson assembly of the plasmid. The other fragments had sufficient amplification with TtrpC containing 82.3 ng/L, noxA containing 51.2 ng/L and PgpdA containing 160.1 ng/L. From the initial PCR protocol mssD was not