Title: Introduction to agrarose gel electrophoresis. Name: D Creaven Date: 4th February 2013 Module: Forensic DNA Laboratory Tutor: Dr Colin Conway Title Introduction to agrarose gel electrophoresis. Date 4th February 2013 Lab Partners Jurgita Baltrukoniene Janet McHugh Objective (a) To prepare the agrarose gel electrophoresis (b) To analysis the movement of DNA on the agrarose gel. Introduction Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs.
Then, the membranes were saturated with 5% (w/v) nonfat milk powder in TBS/T (0.5% Tween 20 in 1× Tris-buffered saline) for 2 h at room temperature. The blots were then incubated overnight at 4°C with primary antibodies, followed by incubation with an IRDye 800CW-conjugated secondary antibody and detection made with LI-COR imaging system (LI-COR Biosciences). The following primary antibodies were used: RELA (BBI, China; 1:1,000 dilution), NFKBIA (BBI, China; 1:1,000 dilution), Lin28B (BBI, China; 1:1,000 dilution), STAT3 (BBI, China; 1:1,000 dilution), RELA (Phospho-Ser276) (BBI, China; 1:1,000 dilution), NFKBIA (Phospho-Ser32/Ser36) (BBI, China; 1:1,000 dilution), STAT3 (Phospho-Ser727) (BBI, China; 1:1,000 dilution), VEGFA (BBI, China; 1:1,000 dilution) and β-actin (Bioworld, USA; 1:5,000 dilution). The immunoreactive bands were quantitated by densitometric scanning. After a wash with TBS/T containing 0.5% Tween 20, the blots with monoclonal antibodies were incubated with peroxidase-conjugated affinipure rabbit anti-goat or goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch Laboratories, USA; 1:4,000 dilution).
1. Denaturing Stage: to separate the double helix, you need to reach about 95 degrees Celsius, then the two strands start to denature. 2. Annealing: In the second stage of PCR, the temp is lowered to about 56 degrees Celsius to allow the primers to “anneal” to then separated strands. A primer is made of a short piece of synthetically made DNA.
2. Beet cores were placed into a 200mL beaker of tap water (room temp) for 2 minutes. After 2 minutes gently removed the beet cores and discarded the rinse water. 3. Placed each beet core into each of six clean dry test tubes.
In Microbiology Lab 3 I chose the unknown culture #14 and ultimately identified it as Staphylococcus epidermidis bacteria. On a microscopic level I found the organism to be gram positive with a coccobacillus shape and both tetrad and cluster cell arrangement. I performed an isolation streak with S. epidermidis on Nutrient Agar which resulted in pinpoint, round, entire, and flat macroscopic morphology. I took a loopful of the organism from the Nutrient Agar and placed it on a slide to perform the catalase test. I added a few drops of 3% Hydrogen Peroxide and it resulted in bubble formation.
GANTC stands for the bases that are found in DNA (Guanine, Adenine, Cytosine and Thymine). The N stands for any nucleotide (phosphorus and sugar). The pieces of DNA are then sorted by size using electrophoresis. DNA particles are placed into an agarose gel and an electrical charge is applied to gel. A positive charge is applied to the bottom and a negative charge to the top of the gel, which is able to conduct electricity.
• Load 1.5mL of the egg white onto the column and collect the eluate in an Eppendorf. Allow the egg white to enter the column. • Add 12 mL of acetate buffer pH 4.6 to the column and collect 1.5mL fractions in Eppendorf tubes. Monitor fractions for protein at A280 and plot A280 against fraction number. If necessary add more acetate buffer pH 4.6 to completely elute the peak.
The suspension is then heated at approximately 95®C for 15-20 minutes. It is then cooled at 4®C and centrifuged, a pellet will form, but the supernatant will be used for the PCR procedure because it contains the DNA. (http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.01_ANTHRAX.pdf). Most reference laboratories use already commercially prepared, that consist of B. anthracis specific primers and probe mixtures that target the genes that code for the virulent toxin. During the PCR reaction, the forward and reverse primers anneal to the complementary sequence on the DNA and a probe that is labelled with a 5’ dye and a 3’ quencher, anneals to the target gene of the bacterium’s DNA.
III. Results and Discussion | 280 nm | 260 nm | 260/280 | 81 AII | 1.444 | 1.291 | 0.89 | 81 BII | 1.450 | 1.255 | 0.87 | 82 AII | 1.483 | 1.383 | 0.93 | 82 BII | 1.466 | 1.296 | 0.88 | Table 1. Absorbances of blood samples in wavelengths 280 and 260 nm with their 260/280 ratios The concentration of DNA in blood samples was known through UV-VIS Spectrophotometer. The spectrophotometer was set to 260 nm and 280 nm. Both wavelengths were used and the absorbance of the blood samples were recorded.
Macromolecule: Nucleic Acid Examples: DNA- Its sugar is deoxyribose; its nitrogenous bases are Adenine, Guanine, Cytosine & Thymine; the number of nucleotides in a molecule is more than 45 million; its shape resembles paired strands coiled in a double helix; and its function is to store genetic information that controls protein synthesis. RNA- Its sugar is ribose; its nitrogenous bases are Adenine, Guanine, Uracil & Cytosine; the number of nucleotides in a molecule is between 100 to 50 thousand; its shape varies with hydrogen binding along the length of the strand with three types: mRNA, tRNA & rRNA; and its function is to perform protein synthesis as directed by DNA. Structure: Nucleic acids contain carbon, oxygen, hydrogen, nitrogen and phosphorus with one or two long chains of nucleotides formed by dehydration synthesis. Each nucleotide has three components covalently bound together: a pentose (a five carbon sugar) attached to both ribose (RNA) and deoxyribose (DNA); a phosphate group; and a nitrogenous base (adenine, guanine, thymine, cytosine & uracil-which replaces thymine in RNA). All nucleic acids have two distinctive ends: the 5’ (5-prime) and 3’ (3-prime) ends, which refers to the carbons on the sugar.