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BIO LAB FINAL
Everything taught throughout the Semester
| Question | Answer |
|---|---|
| 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). |
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