Full Review Flashcards
(283 cards)
1
Q
Biotechnology
A
The use of modern molecular and microbial techniques to make useful products or processes.
2
Q
Vocabulary/Terms
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3
Q
Adherent Cultures
A
So cultures that adhere or stick to a solid surface, such as tissue culture, dish, or flask
4
Q
Insulin
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A hormone regulating blood sugar and commonly used to treat diabetes.
5
Q
Affinity
A
An attraction or likeness to something
6
Q
Enzymes
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Biological catalysts, speeding up chemical reactions in living organisms
7
Q
Chromatography
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A technique used to separate and analyze complex mixtures based on their differential interaction with a stationary phase and a mobile phase.
8
Q
Polyadenylation
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The addition of a poly(A) tail to the 3’ end of eukaryotic mRNA, playing a role in mRNA stability and translation.
9
Q
Release Factor
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A protein that binds to the ribosome when a stop codon is encountered, leading to the termination of protein synthesis.
10
Q
DNA Polymerase
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Enzyme used in PCR to extend the primers by adding complementary nucleotides to the template DNA, usually from thermophilic bacteria
11
Q
Fluorescence
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The emission of light by a substance that has absorbed light or other electromagnetic radiation.
12
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Chapter 1
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The Biotechnology Industry
13
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Genetic Engineering
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Process of manipulating genes
14
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Genomics
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Investigates whole genome, full complement of DNA in cell
15
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Proteomics
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Studies proteome, entire protein complement of cell
16
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Metabolomics
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study of metabolic expression in cells
17
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Microbiomics
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Investigates microbiome, all microorg. living in a place at the same time
18
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Bioinformatics
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Info tech for biology applications. Gather, store, sort and analyze data.
19
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Biosensors
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Biotech instrument that converts biological actions of molecules or organisms to eletricsl signals.
20
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Bioremediation
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Using organisms to convert hazardous waste into less hazardous. Organisms that clean up waste are naturally occurring or genetically engineered.
21
Q
Endangered animals
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Species at risk of extinction due to factors such as poaching, habitat loss, and environmental changes.
22
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Innocence Project
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Non-profit organization using DNA evidence to exonerate wrongfully convicted individuals.
23
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Genetic variability
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Differences in DNA sequences among individuals, used for identification and tracing ancestry.
24
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Genographic Project
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Initiative mapping human migration patterns through DNA analysis.
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DNA profiling
Process of analyzing and identifying individuals based on unique DNA patterns.
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Nanobiotechnology
Application of biological molecules at atomic and molecular scales for specific functions
27
Rexin-G
Nanotechnology-based drug targeting metastasized pancreatic cancer cells.
28
Aquaporin
Protein utilized in artificial membranes for purifying water on a nanoscale.
29
Biotechnology Industry Worth
Expected value to exceed $700 billion by 2025, reflecting its significant economic impact.
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Start-Up Biotechnology Company
Emerging company with innovative teams, often backed by private funding.
31
Chapter 2
Laboratory Safety
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General Product Development Timeline
Conceptualization, Research, Development, Commercialization
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Conceptualization
Market research finds need for product, researchers confirms product can be made. Legal work as well.
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Research
Scientist create initial products, depends on type of product
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Development
Refine product, investigate best manufacturers, detail specifications, testing (safety/effciency)
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Commercialization
Details of marketing manufacturing and distribution decide, preparation for sale
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Orphan Drug Act (ODA)
Incentivizes pharmaceutical companies to develop drugs for rare diseases, which are those affecting fewer than 200,000 people in the United States.
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Quality Assurance Department
Ensures that the company complied with its own quality system and any applicable government regulations. (Requires records be maintained on operations). Makes business dealings smooth and other companies more likely to work with you.
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Good Laboratory Practice
A quality system, a set of regulated practices used to collect safety data on product during development
40
What does GMP stand for?
Good Manufacturing Practices
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What is the purpose of Good Manufacturing Practices?
To ensure steps are taken to produce consistently safe and effective products.
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Return on Investment (ROI)
Financial metric evaluating the gain from an investment relative to its cost, crucial for biotechnology ventures
43
Patent
Exclusive rights granted for a new invention, crucial for protecting biotechnological innovations.
44
Food & Drug Administration (FDA)
Regulatory agency ensuring safety of food, beverages, and therapeutic drugs, vital for biotechnology products
45
Environmental Protection Agency (EPA)
Regulatory agency safeguarding human health and the environment from pollutants and toxic substances, impacting biotechnology practices.
46
United States Department of Agriculture (USDA)
Regulatory agency ensuring protection for agriculture, livestock, and genetically modified organisms, relevant to biotechnology.
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Occupational Safety and Health Administration (OSHA)
Regulatory agency ensuring workplace safety and health for employees, crucial for biotechnology companies
48
National Bioengineered Food Disclosure Law
Legislation requiring guidelines for labeling foods containing genetically modified organisms (GMOs), impacting biotechnology and consumer awareness.
49
Non-GMO Project
Not-for-profit agency providing third-party verification and labeling for foods not containing GMOs, relevant to biotechnology and consumer preferences.
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Animal and Plant Health Inspection Service (APHIS)
Regulatory agency overseeing field testing of genetically modified plants and their impact on agriculture and the environment, crucial for biotechnology and environmental impact assessment
51
Chapter 3
Microbiology and Cell Culture
52
What is a single colony?
A colony from a single bacterium that has multiplied on a solid medium. It's like a round visible dot.
53
What is the purpose of the quadrant method?
Allows isolation of a single colony from a bacterial culture by splitting the plate into quadrants and diluting the bacterium repeatedly as loop a streak through each quadrant.
54
How do serial dilutions allow you to count bacteria?
Serial dilutions allow the number of bacteria to be diluted by tenfold (normally). There will be much less bacterium in the solution, allowing for accountable size of CFU's on the plate.
55
What wavelength of light do you set a spectrophotometer to if you want to detect bacteria?
600nm
56
In gram staining which is the decolorizer?
Alcohol
57
In grim staining what is the color of each stain?
Crystal violet is purple and Safranin is pink
58
Is a bacteria is gram positive
It has a think peptidoglycan layer
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If a bacteria is gram negative
It has a thin peptidoglycan layer
60
Which type of bacteria is more resistant to antibiotics? Why?
Gram-negative because their unique cell wall structure. Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane, which act as a barrier to many antibiotics. This outer membrane contains pores and can actively expel antibiotics, reducing their effectiveness.
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What color is gram positive bacteria?
purple
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What color is gram negative bacteria?
pink
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Chapter 4
DNA Structure and Analysis
64
Recombinant DNA
DNA that has been assembled by linking fragments of DNA from different organisms
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Restriction Enzymes
Bacterial enzymes that cut DNA in very predictable locations
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What are bacteriophages?
Viruses that inject their DNA into bacteria and then use the bacterial cellular machinery to reproduce more copies of themselves.
67
How did restriction enzymes evolve as a defense against bacteriophages?
Restriction enzymes evolved to cut the DNA that was inserted by bacteriophages from the bacterial DNA destroying the phage.
68
How do bacteria protect their own prokaryotic DNA from being lysed restriction enzymes?
The phosphate on the 5' and of the plasma DNA is removed using an enzyme phosphatase.
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What does it mean that the restriction sites tend to be palindromic?
It means that the sites cut from 5' to 3' read the same from 3' to 5'.
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Blunt End
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Sticky End
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After one cut, how many pieces does a plasmid have?
One piece
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How many cuts/ restriction site does there need to be to divide the plasmid into two pieces.
Two cuts
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Ligase
An enzyme that reforms phosphodiester bonds.
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Why might it be easier to connect sticky ends than blunt ends (assuming the sticky ends were cut with the same restriction enzyme)?
The overhang from single each strand would have the complementary stares of the overhang from the other strands. The hydrogen bonds between the two complementary strands would allow for easier joining.
76
What are plasmids?
small extra genomic, circular loops of DNA, found in bacteria
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Why is there a higher probability of the plasma ligating to itself then incorporating a new fragment?
The two ends of the open plasmid are in close proximity to each other while the DNA fragments are floating around the solution.
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How do we stop the DNA from ligating to itself, and allow it to incorporate a new fragment?
The phosphate on the 5' end of the plasma DNA is removed using an enzyme phosphatase. The DNA can only recirculate if the new fragment is added.
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Why are reaction buffers supplied with enzymes?
It ensures that the enzymes are provided with the optimal reaction conditions.
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What is double digest?
When two restriction enzymes are used to digest DNA the same tube.
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What is commonly in a restriction enzyme buffer?
The optimal salts and pH, for that specific enzyme, some also require additives such as bovine serum albumin.(BSA)
82
Why doe enzymes come in glycerol?
Enzyme in glycerol will not freeze at -20C (recommended storing temp). Repeated freeze-thawing of enzyme should be avoided since it reduces enzyme activity.
83
Where should enzymes stay on when actively working in the lab?
Ice or at 4C
84
What substances are included in a typical restriction digest?
Template DNA, Restriction Enzymes, Buffer, BSA(Optional)
85
What does CRISPR stand for?
Clustered Regularly Interspace Short Palindromic Repeats
86
What were all of those sections of spacer DNA in between palindromic repeats likely from?
Viruses/ Bacteriophages when a bacterium or archaeon is infected by a virus, the microbe captures some of the viral DNA and it sorted it into a CRISPR sequence as a space.
87
How will CRISPR spaces be perfect for hybridizing to the viral DNA if it ever shows up again?
Since the crisper sequence is contained in genomic DNA and is passed on to each generation and the library continues to grow. Should a matching virus infect daughter cells descended from this bacterium the spacer RNA transcript and CAs 9 complex would bind and cleave to the viral DNA to prevent it from replicating.
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Cas 1 and Cas 2
Cut and capture the viral DNA.
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The second phase of taking in viral DNA
The spaces are transcribed in each binds to a Cas 9 protein to form a "search and destroy complex" in the cell.
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When introducing new genes by transformation, the genes may insert themselves in a way that disrupts other genes affect their gene expression or cause other unintended effects. This is assuming that a genius directly being placed inside genome (not as a plasmid). Explain where in a gnome, whether encoding or non-coding regions a gene could be placed that might have such impacts.
- Could be placed in the middle of a gene
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-A placing between nucleotides Between that alter the frame reading for codons.
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What makes CRISPER-Cas9 technology so beneficial?
Allow scientist to manipulate genes and gene expression, precisely, and in the least intrusive manner possible.
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Cas 9 enzyme
Endonuclease that cuts double strained DNA at a site dictated by the particular God RNA that is bound to the Cas 9
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Guide RNA (gRNA)
RNA approximately 100 nucleotides long that form a complex with CAs 9. A 20 nucleotide region at the end of the gRNA contains a spacer sequence complementary to the target DNA sequence.
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How does CRISPR utilize cellular machinery?
The cell recognizes the DNA is damaged and
96
starts to repair it. Scientist take advantage of the ways that cellular DNA repair machinery works to introduce changes to one or more genes.
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Using CRISPR on somatic/body cells only affects the individuals who you do it on. Why might people find it ethically questionable to use gametes or germ line cells?
With gametes the CRISPR mutation would get passed on to that organisms or persons offspring.
98
Why are combs placed on the side closest to the negative electrode?
DNA fragments are negatively charged. They repel from the negative electrodes and are attracted to the positive.
99
What are the most common electrophoresis buffers?
Tris/acetic acid/EDTA (TAE) & Tris/boric acid/EDTA (TBE)
100
What are DNA size standards?
Sample that contains DNA fragments of known sizes for comparison of unknown fragments.
101
How are DNA size standards made?
Made from bacteriophage or plasma DNA cut with specific restriction enzymes that yield DNA fragments of known number size
102
How are rulers/standards named?
Rulers are named based off the smallest fragment, located on the ruler.
103
What aspects of the band can tell us the quantity of DNA how can we take advantage of that from a standard?
The intensity or thickness of the DNA band, having the known band size from a standard. The thickness of the unknown band can be compared to the standard to estimate the quantity.
104
Recombinant DNA Technology
A set of molecular techniques for creating recombinant DNA molecules, including cloning, gene manipulation, and expression of foreign genes in host organisms, essential for biotechnology and genetic engineering.
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Restriction Digest
The process of cutting DNA at specific sequences using restriction enzymes, which is crucial for genetic engineering and DNA manipulation.
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Double Digest
The simultaneous digestion of DNA with two different restriction enzymes, allowing for more precise DNA manipulation and analysis.
107
Agarose Gel Electrophoresis
A fundamental technique for separating DNA fragments based on size using an electric field in agarose gel, essential for DNA analysis and profiling.
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DNA Profiling
The analysis of DNA fragments to create a unique genetic fingerprint for each individual, widely used in forensic investigations and genetic studies.
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RFLP
Restriction Fragment Length Polymorphisms - genetic variations in DNA sequences due to mutations in restriction sites, important for genetic diversity and mutation analysis. Certain mutations in restriction sites make it so that the sites are no longer recognized by the restriction enzyme.
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How can RFLPs be used to identify an individual?
The unique, genetic differences would be unique to that person so analyzing the RFLP can help you identify a person
111
Southern Blotting
A technique to detect specific DNA fragments in a sample after agarose gel electrophoresis, commonly used for DNA analysis and genetic research. The DNA fragments are transferred out of the cellular matrix onto a solid membrane, which is then exposed to a DNA pro labeled with radioactive, fluorescent, or chemical tags. Any DNA fragments containing complementary sequences with the DNA probe will be visualized.
112
Northern Blot
A laboratory analysis method used to study RNA by separating them based in size utilizing electrophoresis. The RNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to DNA probably with a radioactive or chemical tag.
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Western Blot
A laboratory technique used to detect a specific protein in a blood or tissue sample to the means of gel letters, the separated proteins I transferred out of the gel to the surface of a membrane. The membrane is exposed to an antibody specific to the target protein.
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Eastern Blotting
A technique used to analyze protein, post transitional modification, such as glycation, phosphorylation, and lipidation. An extension of western plotting, but instead of detecting the presence of specific proteins, it detect specific modifications to those proteins. The probes bind to transitional modifications on proteins.
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DNA Staining
The process of visualizing DNA in agarose gel using dyes or fluorescent stains, essential for DNA analysis and visualization.
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Why can positively charged stains be used with DNA?
Positively charged stains with bond with a negatively charged DNA fragments.
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Why must we use the positively charged stains after the gel has been run?
If not, the dice would disrupt the migration of the DNA in the gel.
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Some stains are not positively charged, and so they can be added to the sample before running the gel. Examples of these are SYBR Safe DNA Stain, Gel Red, UView 6X loading dye, and ethidium bromide. How do these attach the DNA if they are not positively charged?
The dyes fit between the base pairs of DNA.
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Ethidium Bromide
A common DNA stain used in research and industry, but is a known mutagen and possible carcinogen, caution is advised when handling.
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SYBR Safe DNA Stain
A safer alternative to ethidium bromide for staining DNA in agarose gel, ensuring safety in DNA analysis and research.
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Forensic Evidence
Material containing DNA used in criminal investigations to identify suspects or victims, crucial for forensic science and law enforcement.
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Chapter 5
Bacterial Transformation and Plasma Purification
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Chapter 6
The Polymerase Chain Reaction
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Polymerase Chain Reaction (PCR)
A technique to make multiple copies of a specific DNA segment. Simplifies version of bacterial DNA replication that copies a specific sequence to amplify it. Copies replicated over and over again.
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What are primers in the context of DNA?
Short single strands of DNA that target a specific sequence.
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What is the purpose of primers?
Designed to match and bind to each end of the target sequence.
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Forward Primers
The first primer that anneals at the beginning of the targeted region of DNA
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Reverse Primer
The 2nd primer designed to bind at the end of the targeted region of the DNA strand.
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3 Stages of PCR:
Denaturation, Annealing, and Extension
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Denaturation
First stage of PCR, where the DNA double helix is separated by heating to 94°C, making it single-stranded.
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Annealing
What is the second stage of PCR
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What happens during the annealing stage of PCR?
Primers bind to the single-stranded DNA at a specific temperature.
133
What allows hydrogen bonds to form during the annealing stage of PCR?
Cooled to 50-60°C to allow the PCR mixture bonds to form between primer and single-stranded DNA template.
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Extension
Third stage of PCR at 72C where DNA polymerase extends the primers by adding nucleotides to the 3' end, creating new DNA strands.
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Thermal Cycler
Instrument used to amplify DNA segments through repeated cycles of heating and cooling, essential for PCR.
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Master Mix
A premixed solution containing DNA polymerase, dNTPs, primers, and reaction buffer, ensuring uniform concentrations in PCR reactions.
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Taq DNA Polymerase
A thermostable DNA polymerase enzyme commonly used in PCR.
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EvaGreen* Dye
A DNA-binding dye used in real-time PCR to detect the presence of DNA.
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Real-Time PCR
Quantitative PCR (qPCR), amount of PCR product is measured as each cycle is completed real time & used to deduce the amount of input DNA.
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Digital PCR
Builds off PCR but allows for more sensitive detection and direct measurement of nucleic acid concentration w/ out the use of standard curves
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Reverse Transcription PCR (RT-PCR)
Uses retroviral enzyme reverse transcriptase to reverse transcribe mRNA into DNA before PCR begins. Resulting DNA is used ad a template for PCR.
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Multiplex PCR
Simultaneously detects multiple target sequences on the same starting material in the same reaction tube. Multiple sets of primers each target and amplify a different sequence.
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Degenerate PCR
Amplify DNA when the info about the target sequence is limited or when the sample PCR needs to work with DNA templates from different copies. Primers have same sequence as each other differing 1-3 bases for diff. in sequence of target.
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Nested PCR
Uses 2nd round of PCR & a 2nd set of primers that binds with in the PCR product produced during initial round. Ensures product generated is specific. (When PCR conditions suboptimal, nested used).
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Fast PCR
Total run for 3-cycles of PCR, is 2.5-4 hrs. Using specifically designed primers and 2 step PCR program.
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Isothermal PCR
At a single temperature instead of 3. Faster. Loop mediated isothermal amplification (LAMP) req. multiple sets of carefully designed primers and a polymerase that displaces a stand of DNA as it polymerizes.
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Random Amplification of Polymorphic DNA (RAPD)
Used when the genomic sequence if the DNA is unknown. RAPD identifies differences among similar genomes & is often used to differentiate closely related plant species or bacterial stains. In RAPD PCR, mix of random primers is used to amplify the template for DNA then compared.
148
Chapter 7
Protein Structure and Analysis
149
Proteins
Biological macromolecules composed of amino acids, with diverse functions such as enzymes, hormones, and antibodies.
150
Amino Acids
Organic compounds serving as the building blocks of proteins, each with a unique side chain (R group).
151
Isoelectric Point (pl)
The pH at which a molecule carries no net electrical charge, meaning it is electrically neutral. At this point, the positive and negative charges within the molecule are balanced. The isoelectric point is significant for proteins and amino acids, as it influences their solubility and stability in different pH environments.
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Polyacrylamide Gel Electrophoresis (PAGE)
A technique to separate proteins based on size using a polyacrylamide gel and an electric field, commonly used in biochemistry and molecular biology research.
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Stacking Gel
The portion of a discontinuous polyacrylamide gel with a low percentage of acrylamide that helps concentrate proteins into tight, focused bands for subsequent separation in electrophoresis.
154
Gradient Gel
A polyacrylamide gel with a continuously increasing percentage of acrylamide, allowing the separation of small and large proteins in the same gel, commonly used in protein analysis.
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Separating Gel
The portion of a discontinuous polyacrylamide gel with a high percentage of acrylamide that separates proteins based on size in electrophoresis.
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Cathode
The negatively charged electrode in an electrophoresis system, attracting positively charged ions or molecules, crucial for the movement of biomolecules during electrophoresis.
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Anode
The positively charged electrode in an electrophoresis system, attracting negatively charged ions or molecules, essential for the movement of biomolecules during electrophoresis.
158
Stain-Free Gel
A type of gel used in SDS-PAGE that contains reagents to modify tryptophan amino acids, causing protein bands to fluoresce and be visible without the need for staining, facilitating protein analysis.
159
Native PAGE
A technique to separate proteins based on their net charge to mass ratio rather than their mass alone, without denaturing the proteins, commonly used for analyzing native protein structures.
160
Isoelectric Focusing (IEF)
A technique to separate proteins based on their isoelectric point (pI), the point at which they have no overall charge, widely used in protein purification and analysis.
161
Two-Dimensional PAGE
A technique that separates proteins by their pI (isoelectric point) in one dimension and by their size in another, allowing for high-resolution separation and identification of proteins, commonly used in proteomics research.
162
Size Exclusion Chromatography
A chromatographic technique that separates molecules based on their size, with smaller molecules entering the beads and traveling slowly through the column, widely used for protein purification and analysis.
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Ion Exchange Chromatography
A chromatographic technique that separates molecules based on their net charges, with cation exchange resin binding positively charged molecules and anion exchange resin binding negatively charged molecules, commonly used for protein purification and analysis.
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Hydrophobic Interaction Chromatography (HIC)
A chromatographic technique that separates proteins based on their hydrophobicity, with hydrophobic regions sticking to the resin in high salt conditions, widely used for protein purification and analysis.
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Affinity Chromatography
A chromatographic technique that separates molecules based on their affinity for a specific binding partner, allowing for highly specific purification of proteins, commonly used for protein purification and analysis.
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Bioreactor
A vessel or system for growing cells or microorganisms under controlled conditions, often used for large-scale production of proteins, crucial for biopharmaceutical and biotechnology industries.
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Quality Control (QC)
The set of procedures and tests used to ensure that a product meets specified requirements and standards, including monitoring the production process and testing the final product, essential for ensuring the quality of protein-based products.
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Batch Record
A document that includes instructions and places to note the details of each step during the production of a batch of product, used for tracking and quality assurance in protein production.
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Protein Data Bank (PDB)
A repository for 3-D structural data of large biological molecules, including proteins and nucleic acids, used by researchers to understand protein structures and functions, vital for structural biology and drug discovery.
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Bioinformatics
The application of computer technology to the management of biological information, allowing for the analysis of protein sequences, structures, and functions, crucial for understanding and interpreting biological data.
171
Why should Polyacrylamide gels be used for DNA fragments of less than 1kb?
Polyacrylamide gel is a much tighter matrix so it is able to separate smaller fragments.
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Mobile Phase
The liquid that contains the protein sample, it flows over the stationary phase.
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Stationary Phase
The resin beads/solid part of the chromatography
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Unlike gel electrophoresis, size exclusion chromatography leads small molecules to move slower than larger molecules, how does it do that?
The resin beads contain very tiny holes that small molecules can flow through and large molecules just passover. This means that larger molecules will flow through faster while smaller one take longe4r to flow through.
175
What is the stationary phase in Hydrophobic Interaction Chromatography?
Hydrophobic resin
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What is the mobile phase in Hydrophobic Interaction Chromatography?
The liquid with a high salt concentration.
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Why does hydrophobic regions bind to the resin when initially added as part of a highly concentrated salt solution?
The hydrophobic regions bind to the resin to reduce their exposure to the high salt/highly ionic solution.
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Each round in hydrophobic interaction chromatography a slightly less concentrated salt solution to "elute" the protein form the resin. Explain what this means, and how this works to separate out different proteins by their range of hydrophobicity.
To elute the solution is to release the proteins from the beads. Solutions with a less salt concentration means there is a less ionic environment. So, proteins that are less hydrophobic will be released from the beads as the concentration lowers. As different fractions flow out, they move from a less hydrophobic to more hydrophobic.
179
What charge does a cation exchange resin have?
Negative Charge
180
What molecules does cation exchange resin bind to?
Molecules that are positively charged
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What charge does an anion exchange resin have?
Positive
182
What molecules does anion exchange resin bind to?
Molecules that are negatively charged
183
What type of resin would you run DNA through to purify it?
Anion exchange resin.
184
What would you use to elute ion exchange chromatography?
Buffers with gradually increasing salt concentrations.
185
Recombinant Proteins
Proteins created through genetic engineering for medical treatment.
186
Recombinant tissue plasminogen activator (tPA)
A genetically engineered protein used to dissolve brain clots, improving stroke survival.
187
Collagen Spores
Animal-derived sponges for healing severe burns and ulcers.
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Protein Quantitation
Measuring protein concentration using dyes and a spectrophotometer.
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Protein Quantification
The process of determining the amount of protein present in a sample, often done through various analytical techniques.
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Protein Mass Spectrometry
A method for determining protein size, sequence, structure, and modifications.
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What is a Discontinuous Buffer System?
A Discontinuous Buffer System uses a gel separated into two sections: a large pore stacking gel on top of a small pore resolving gel.
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How do proteins behave in a Discontinuous Buffer System?
Proteins migrate quickly through the large pore stacking gel and are slowed as they enter the small pore resolving gel.
193
What is the effect of proteins stacking in a Discontinuous Buffer System?
Proteins stack on top of one another to form a tight band, which helps improve resolution.
194
How do Discontinuous Buffer Systems compare to Continuous Systems?
Discontinuous Buffer Systems provide higher resolution than Continuous Systems.
195
What is the benefit of varying the buffers in a Discontinuous Buffer System?
Varying the buffers used in the sample, gel, and electrode chambers creates a variety of discontinuous buffer systems for different applications.
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Coomassie Brilliant Blue
A protein stain for visualizing proteins in electrophoresis.
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Fluorescent Stains
Stains reacting with specific amino acid sites on proteins for visualization
198
Lowry Assay
Measures how proteins that have been treated with copper interact with a reagent called Folin-Ciocalteu reagent. The Interaction causes a color change that absorbs light at 750nm.
199
What dye is used in the Bradford Assay?
Coomassie Brilliant Blue G-250
200
What color is the Bradford reagent in the absence of protein?
Reddish-brown
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What is the maximum absorption wavelength of the Bradford reagent without protein?
470nm
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What color does Coomassie Brilliant Blue G-250 turn when it interacts with protein?
Blue
203
What is the peak absorption wavelength of the Bradford reagent when it interacts with protein?
595nm
204
How does the intensity of blue color in the Bradford Assay relate to protein concentration?
The more protein, the more intense the blue color.
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How does the protocol of the Bradford Assay compare to the Lowry assay?
The Bradford Assay uses a quicker and simpler protocol than the Lowry assay.
206
What levels of sensitivity do the Bradford Assay and Lowry assay offer?
Similar levels of sensitivity.
207
What is a batch record? Why is it used at every step of the manufacturing and testing process for a given batch?
A batch record is a document that manufacturing personnel receive to record relevant data during production. It's used at every step so that QA can review all documentation to determine if the product was made and documented correctly.
208
What specifically does a batch record include:
Instructions and places to record relevant info. it has a unique ID # by the QA department that correlates with all test samples taken during production run.
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Chapter 8
Immunology Application
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Immunity
The resistance of an organism to infection or disease and an immune response is triggered by the presence of something foreign to the body.
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Innate Immunity
Natural immunology defenses that protect against pathogens and are present at birth.
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Acquired (adaptive) Immunity
A response to a specific form substance. Some immune cells can adapt so that they are able to remember and recognize specific invaders.
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Immunization
The initial contact with the invader. Begins cascade of events.
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What are antigens?
Factors capable of eliciting an immune response.
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What are some examples of possible antigens?
Microorganisms, microbial products (toxins), foreign proteins, DNA and RNA, drugs, and other chemicals.
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Epitope
Small, accessible portion of an antigen that can be recognized.
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Antibodies
Proteins produced by the immune system in response to specific pathogens, aiding in their neutralization and elimination.
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ELISA
Enzyme-linked immunosorbent assay used to detect and quantify the presence of antibodies or antigens in a sample.
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Serum Antibody Detection
The process of identifying specific antibodies in a blood sample, crucial in diagnosing infectious diseases and immune disorders.
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Quantitative ELISA
An ELISA used to measure the amount of a specific protein or antibody, providing quantitative data in research and diagnostics.
221
Macrophage
A type of cell that removes foreign cells and molecules from the blood by phagocytosis (innate immunity) and their process antigens and present them on their cell surface (acquired immunity).
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Pathogen
An organism that can cause disease, including bacteria, viruses, fungi, and parasites.
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B Cell
A type of white blood cell that produces antibodies. B cells mature in the bone marrow. They present antigenic epitopes on their surface to attract T cells.
224
Why is there a huge number and diversity of antibodies?
B cells have the ability to rearrange their DNA to make different antibody genes
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T Cell
Stimulate the proliferation of B cells that have bound to an antigen, and they kill whole cells that are infected by a virus to prevent the virus from infecting other cells
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Plasma Cell
A type of white blood cell that produces and secretes antibodies, vital for the body's adaptive immune response.
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Immunoglobins
another word for antibodies
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Types of Immunoglobulins
IgG, IgA, IgM, IgD, IgE
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Which immunoglibon is utilized most in biotechnology?
IgG
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Major Histocompatibility Complex (MHC)
A group of genes that code for proteins found on the surfaces of cells, crucial for immune recognition and self-versus-nonself discrimination. Macrophages present antigenic epitopes on their cell surfaces to be recognized by T cells using the MHC.
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Polyclonal Antibodies
Antibodies produced by different B cell clones in response to an antigen, used in various research and diagnostic applications.
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Pros and Cons of Polyclonal Antibodies
Pro: simple and inexpensive to produce
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Con: no two batches, even though made in the same animal are exactly the same
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What are monoclonal antibodies?
Antibodies produced by identical immune cells that are clones of a unique parent B cell.
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What are monoclonal antibodies used for?
They are widely used in targeted therapy and diagnostics.
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Humanized Monoclonal Antibodies
Monoclonal antibodies engineered to contain human protein sequences, reducing immunogenicity and enhancing therapeutic efficacy.
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Hybridoma
A cell resulting from the fusion of a cancer cell with a B, used for large-scale production of monoclonal antibodies since specific B cells is produced over and over indefinitely.
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Fusion Proteins
Combine genes from different organisms to produce a protein product that contains sequences from both species.
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What is a humanized antibody?
A human antibody that has been engineered to contain some of the antigen binding region (Fab) of a mouse monoclonal antibody.
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What provides the specificity for a humanized antibody?
The mouse protein sequence.
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Why does the human portion of a humanized antibody not evoke an immune response?
Because it is familiar to the body.
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XenoMouse
A transgenic system in which the genes for human antibodies have been cloned into a line of mice so that when the mice are immunized, they produce human antibodies rather than mouse antibodies from their cells B cells
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Human Combinatorial Antibody Libraries (HuCAL)
A compilation of more than 45 billion synthetically designed functional human antibody genes cloned into a stage library. And in vitro selection technology can be used to select genes from the library that encode for antibodies that bind to almost any antigen.
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Immunoassays
Tests that use antibodies to detect or measure molecules in samples, such as ELISAs and dipstick tests
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Quantitative Immunoassays
Methods for determining the amount of a particular substance using immunoassays
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Agdia Immunoassays Kit
Kit for quantifying the amount of transgenic proteins in agricultural products using immunoassays
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Enzyme Immunoassays (EIA)
Tests performed in microplates or tubes, such as ELISAs, to detect or measure molecules using antibodies
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Ouchterlony Assays
Immunoassays using agarose gels for double diffusion tests to detect or measure molecules
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Immunohistological Assays
Tests performed on tissue or cell samples on microscope slides to detect specific proteins utilizing antibodies.
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High-throughput Assays
Assays utilizing microplates and microscopic beads, such as multiplex bead assays, for detecting or measuring molecules
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Fluorescence-Activated Cell Sorting
Assay performed on whole cells for high-throughput analysis using antibodies to detect specific proteins
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Primary Antibody
Antibody binding to the antigen of interest
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Secondary Antibody
Antibody recognizing the primary antibody
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Monoclonal Antibody
Antibody binding to a single epitope on the molecule of interest, used in immunoassays to detect proteins
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polyclonal antibody
Antibody binding to many different epitopes on the target, used in immunoassays to detect or measure molecules
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Direct ELISA
ELISA using a primary antibody labeled w/ an enzyme for direct antigen detection
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Indirect ELISA
ELISA using both primary and enzyme-linked secondary antibodies for indirect antigen detection
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Sandwich ELISA
A type of ELISA assay that detects and quantifies antigens by "sandwiching" them between two antibodies
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Electroblotting
Technique using an electric current to transfer proteins from a gel to a membrane, commonly used in Western Blots
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Microplate Reader
Instrument measuring the absorbance of samples in microplates, used in various immunoassays
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Chromogenic Substrate
Substance generating a color at a specific wavelength, commonly used in ELISA
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Semi-dry Blotter
Apparatus performing electroblotting in a few minutes compared to traditional methods, used in Western Blots
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Immunodetection
Process involving incubation with primary and secondary antibodies to detect specific proteins, a key step in immunoassays
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Math/Calculations
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Calculate the concentration of DNA in a plasmid prep that when tested at 270nm gave an absorbance of 0.85au.
42.5 ug/uL
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2-step 9-fold dilution, what's the dilution factor?
1/81
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9-step 2-fold dilution
1/512
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5-step 6-fold dilution
1/7776
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4-step 4-fold dilution
1/256
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Formula for calculating total amount of colonies after dilution
(Dilution Factor * Number of Colonies Counted)/ volume of culture pippeted
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Extra Info
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PNA
Peptide nucleic acid, a synthetic nucleic acid analog that can bind to DNA and RNA.
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Inventory
A detailed list of all components and conditions specific to a PCR reaction.
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Labs
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Tratuzumab
A humanized monoclonal antibody used to treat cancer, specifically targeting HER2-positive breast cancer and gastric cancer.
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Rituxumab
A monoclonal antibody used to treat non-Hodgkin's lymphoma and rheumatoid arthritis, targeting CD20-positive B cells.
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Human Chorionic Gonadotropin
Hormone indicating pregnancy, detected in blood via immunoassays like pregnancy tests
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SYBR Green Supermix
A reagent mix used in real-time PCR to detect and quantify DNA.
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Why is it important that you remove the pipet tip from the buffer before releasing the plunger?
Releasing the plunger will cause the sample and the buffer to be sucked back into the pipet.
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What do you mix to make agarose gels?
Mixing agarose powder with 1x electrophoresis buffer
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What percentage of agarose is used for more than 1000 bases (1kb)?
0.7-1%(m/v)
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Why is it important that you "slowly" press down on the pipet plunger when releasing the sample from the pipet tip?
Both the sample and the buffer are liquids, pressing slowly ensures that the sample properly gets/flows down into the well if not, the sample results could be inaccurate.
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What are the two things are you using your buffer for?
The buffer is used to make the gel and is used to fill the chamber.
