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Everything taught throughout the Semester

What are antibodies? proteins that can bind to molecules that “appear” foreign and/or are associated with a dangerous threat, namely a pathogenic organism.
What is an antigen? foreign protein
How is HIV spelled out? Human Immunodeficiency Virus
How is ELISA spelled out? Enzyme-Linked Immunosorbent Assay
What happens in an ELISA? he antibodies would be able to detect the presence or absence of the HIV protein in a patient’s blood sample.
What is p24? is a viral capsid protein, and there are roughly 2000 copies of this particular protein within each virus particle.
How is p24 released? When a person is infected with HIV and the virus is lysed (broken open and destroyed), as part of an infected individual’s immune defense
What does a positive ELISA test result mean? indicates that the patient does possess p24 and is infected with HIV
How does an ELISA work? There must be a way to detect antibody interaction with the antigen
What is the first coating on the solid support? Antigen
What does the secondary antibody consist of? tagged with an enzyme that will have the ability to convert a substrate into a colored product that can be visualized.
How do you spell out PCR? polymerase chain reaction
What does PCR Produce? exponentially large amounts of a specific DNA from trace amounts of starting material (template).
What is a genotype? described as a particular set of genetic markers, or alleles, in a DNA sample.
How many basepairs are in the human genome? approximately 3 billion basepairs
What is loci? Many of the DNA sequences used in forensic profiling come from regions of our chromosomes
non-coding regions possess what? short tandem repeats
What are STRs? very short DNA sequences that are repeated in direct head-to-tail fashion
alleles in a population a single locus may have different forms or types
How many alleles of TH01 do we have? 2; located on chromosome #11
Can alleles of TH01 be identical or non-identical? both
heterozygous one with 5 TCAT repeats and one with 3 TCAT repeats
homozygous possesses two TH01 alleles each with 5 TCAT repeats.
What happens after PCR is completed? the amplified products will be analyzed using agarose gel electrophoresis
What is electrophoresis? the separation of molecules, on a solid support, in an electric field; agarose provides a solid support for the movement of DNA molecules during electrophoresis.
what does electrophoresis seperates? DNA molecules (fragments) according to their relative size
___molecules can move through the agarose matrix more easily than larger ones smaller
The Protein Expression and Purification Series focuses on the protein ___________. dihydrofolate reductase (DHFR)
What is dihydrofolate reductase (DHFR) is essential for? proper cell function and illustrates the importance of basic oxidation– reduction enzymatic reactions
What is DHFR? an enzyme that converts dihydrofolate, a folic acid derivative, into tetrahydrofolate (THF) by the addition of a hydride from NADPH.
What does pET21a plasmid contain? the constitutively expressed β-lactamase gene that confers resistance to ampicillin.
Day 1: Culturing E.Coli for Protein Expression lyophylized BL21(DE3) E. coli containing pDHFR which will be rehydrated and plated to generate individual colonies.
Day 2: Culturing E.Coli for Protein Expression An initial culture is grown to saturation from a single bacterial colony picked from a Petri dish.
Day 3: Culturing E.Coli for Protein Expression culture is used to initiate a larger culture that is grown to mid-log phase at which stage expression of the recombinant protein is induced by addition of IPTG to the medium.
Day 4: Culturing E.Coli for Protein Expression After induction has been completed, cells are recovered by centrifugation, and protein is extracted from the cell pellet.
Day 5: Protein Purification Once the cells are lysed, the soluble and insoluble components are separated by centrifugation
Day 6 & 7: Isolating Recombinant Protein Proteins are usually purified by chromatography.
What is a buffer? The liquid used to dissolve the biomolecules to make the mobile phase
What is a sample? the mixture of biomolecules dissolved in the buffer
purification of the GST-DHFR-His -First the protein will be purified using IMAC. -removing excess salt and imidazole from the sample
What does chromatography allow? the separation of individual components from complex mixtures.
different types of chromatography Thin layer chromatography, Gas chromatography, Ion exchange chromatography, Size exclusion chromatography
Hemoglobin 65,000 Da – large protein with a red/brown color.
Vitamin B12 1350 Da – small molecule with a pink color.
Which molecule eluted first? Hemoglobin
gene a segment of DNA that provides the instructions for the production of a protein product that leads to a particular trait in an organism.
What does Genetic Transformation involve? the insertion of a gene (or genes) into an organism in order to change the organism's traits
bacterium -it is a prokaryote with no nucleus and no membrane bound organelles. -In order to transform bacteria we will use plasmids.
plasmid double stranded circular DNA molecule that can exist extra chromosomally in bacteria
pGLO plasmids contain 3 genes 1.GFP producing gene 2. bla gene 3.araC gene
Green Fluorescent Protein -gene from jelly fish -GFP can be seen using UV light. - transformation can be seen in real time
bla (Beta-lactamase) -bla gene provides instructions to make Beta-lactamase protein -protein/enzyme provides antibiotic resistance by breaking down the antibiotic, ampicillin.
araC - gene that provides instructions to make araC protein and helps to turn-on the GFP gene in the presence of sugar arabinose
arabinose precense -araC gene gets activated -araC protein is produced -Activates GFP gene -Production of GFP (glow)
no arabinose -araC gene is not activated -araC protein isn’t produced -GFP gene is not activated -NO GFP . NO glow
LB Plate (-pGLO) -growth -This is a control to check e.coli viability after transformation process.
LB +Amp (-pGLO) This is a control to check that ampicillin works. No growth b/c of the amp.
LB + Amp (+pGLO) -Growth -Transformed cells produce beta-lactamase and live on amp plates. Non- transformed cells will die.
LB + Amp + Arabinose (+pGLO) -Growth & Glow -Good transformation. Arbinsose turned on the araC gene which turns on GFP gene.
two types of cells (i) suspension cells that comprise all of the blood cells (red, white and megakaryocytes), and (ii) adherent cells (every other body cell type).
Created by: savepeople
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