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Questions and Answers
Questions and Answers
Which of the following best describes the primary function of DNA?
Which of the following best describes the primary function of DNA?
- Providing structural support to cells
- Catalyzing biochemical reactions
- Storing and transferring genetic information (correct)
- Regulating gene expression
What is the key difference between D-ribose and 2-deoxy-D-ribose?
What is the key difference between D-ribose and 2-deoxy-D-ribose?
- 2-deoxy-D-ribose lacks an oxygen atom at the 2' carbon. (correct)
- D-ribose contains a phosphate group, which is absent in 2-deoxy-D-ribose.
- 2-deoxy-D-ribose is only found in RNA, while D-ribose is found in DNA.
- D-ribose is a pentose sugar, while 2-deoxy-D-ribose is a hexose.
Which of the following nitrogenous bases is a purine?
Which of the following nitrogenous bases is a purine?
- Thymine
- Uracil
- Guanine (correct)
- Cytosine
According to the Brønsted-Lowry definition, what characteristic defines a base?
According to the Brønsted-Lowry definition, what characteristic defines a base?
What type of chemical bond is formed by a phosphate group linking two sugar residues in a strand of nucleic acid?
What type of chemical bond is formed by a phosphate group linking two sugar residues in a strand of nucleic acid?
What is the composition of a nucleoside?
What is the composition of a nucleoside?
What chemical component, when added to a nucleoside, forms a nucleotide?
What chemical component, when added to a nucleoside, forms a nucleotide?
In the structure of DNA, what type of bonds connect complementary base pairs?
In the structure of DNA, what type of bonds connect complementary base pairs?
What is the significance of the 5'-to-3' and 3'-to-5' orientation of DNA strands in a double helix?
What is the significance of the 5'-to-3' and 3'-to-5' orientation of DNA strands in a double helix?
Which series of steps is used in nucleic acid isolation?
Which series of steps is used in nucleic acid isolation?
Why is it important to use RNase-free tubes and reagents during RNA isolation?
Why is it important to use RNase-free tubes and reagents during RNA isolation?
Which of the following is a characteristic of the organic method (Phenol: Chloroform) of nucleic acid extraction?
Which of the following is a characteristic of the organic method (Phenol: Chloroform) of nucleic acid extraction?
What is the purpose of using saturated NaCl in the inorganic method of DNA extraction?
What is the purpose of using saturated NaCl in the inorganic method of DNA extraction?
Which of the following best describes the advantage of using a solid-phase method for DNA isolation?
Which of the following best describes the advantage of using a solid-phase method for DNA isolation?
In Chelex extraction, what happens to the contaminating substances during the process?
In Chelex extraction, what happens to the contaminating substances during the process?
What is the purpose of Tris-EDTA in DNA extraction protocols?
What is the purpose of Tris-EDTA in DNA extraction protocols?
What wavelength is typically used to measure the concentration of DNA in spectrophotometry?
What wavelength is typically used to measure the concentration of DNA in spectrophotometry?
A sample of dsDNA is diluted 100X, and the diluted sample gives a reading of 0.45 on a spectrophotometer at OD260. What is the concentration of DNA in the original sample?
A sample of dsDNA is diluted 100X, and the diluted sample gives a reading of 0.45 on a spectrophotometer at OD260. What is the concentration of DNA in the original sample?
What does an A260/A280 ratio of less than 1.8 indicate about a DNA sample?
What does an A260/A280 ratio of less than 1.8 indicate about a DNA sample?
What is the primary role of primers in the polymerase chain reaction (PCR)?
What is the primary role of primers in the polymerase chain reaction (PCR)?
In PCR, what happens during the annealing step?
In PCR, what happens during the annealing step?
What is the purpose of using a thermostable DNA polymerase, such as Taq polymerase, in PCR?
What is the purpose of using a thermostable DNA polymerase, such as Taq polymerase, in PCR?
Which of the following is a condition for primer design?
Which of the following is a condition for primer design?
What is the function of MgCl2 in PCR?
What is the function of MgCl2 in PCR?
Which type of control is used to ensure that the PCR reaction mix is not contaminated with template DNA or amplified products from previous runs?
Which type of control is used to ensure that the PCR reaction mix is not contaminated with template DNA or amplified products from previous runs?
What is the purpose of ethidium bromide in agarose gel electrophoresis?
What is the purpose of ethidium bromide in agarose gel electrophoresis?
During agarose gel electrophoresis, which DNA fragments migrate faster?
During agarose gel electrophoresis, which DNA fragments migrate faster?
During gel electrophoresis, supercoiled DNA migrates at different rates than linear or circular DNA. Which of the following is true?
During gel electrophoresis, supercoiled DNA migrates at different rates than linear or circular DNA. Which of the following is true?
A researcher loads a DNA ladder onto an agarose gel. What is the primary purpose of the DNA ladder?
A researcher loads a DNA ladder onto an agarose gel. What is the primary purpose of the DNA ladder?
Which of the following best explains how real-time PCR monitors the amplification of a targeted DNA molecule?
Which of the following best explains how real-time PCR monitors the amplification of a targeted DNA molecule?
What is the key difference between non-specific dyes and sequence-specific probes in real-time PCR?
What is the key difference between non-specific dyes and sequence-specific probes in real-time PCR?
What is the purpose of the quencher in a TaqMan probe used in real-time PCR?
What is the purpose of the quencher in a TaqMan probe used in real-time PCR?
What is the function of reverse transcriptase in reverse transcription PCR (RT-PCR)?
What is the function of reverse transcriptase in reverse transcription PCR (RT-PCR)?
What is a key advantage of dPCR (digital PCR) compared to traditional PCR and qPCR?
What is a key advantage of dPCR (digital PCR) compared to traditional PCR and qPCR?
What is the purpose of denaturing a double-stranded target DNA after electrophoresis and prior to transfer in a Southern blot?
What is the purpose of denaturing a double-stranded target DNA after electrophoresis and prior to transfer in a Southern blot?
What is the primary application of Northern blotting?
What is the primary application of Northern blotting?
In Western blotting, what is the purpose of using a secondary antibody?
In Western blotting, what is the purpose of using a secondary antibody?
What is a general limitation to molecular testing?
What is a general limitation to molecular testing?
Questions and Answers
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Flashcards
Flashcards
Nucleic Acid
Nucleic Acid
Polymeric macromolecules made of repeated monomeric nucleotide units.
DNA (Deoxyribonucleic Acid)
DNA (Deoxyribonucleic Acid)
A polymer of deoxyribonucleotides that stores and transfers genetic information.
RNA (Ribonucleic Acid)
RNA (Ribonucleic Acid)
A polymer of ribonucleotides involved in protein synthesis and gene expression regulation.
Nucleotide
Nucleotide
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Pentose Sugars
Pentose Sugars
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Bronsted-Lowry base
Bronsted-Lowry base
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Lewis Base
Lewis Base
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Arrhenius Base
Arrhenius Base
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Phosphate Group
Phosphate Group
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Nucleoside
Nucleoside
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Nucleotide
Nucleotide
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Base Pairs
Base Pairs
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DNA and RNA Isolation
DNA and RNA Isolation
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Cell Lysis
Cell Lysis
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Extraction
Extraction
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Purification
Purification
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Nucleic acid extraction
Nucleic acid extraction
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Spectrophotometry
Spectrophotometry
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Target Amplification
Target Amplification
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Signal Amplification
Signal Amplification
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Probe Amplification
Probe Amplification
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Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
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Taq Polymerase
Taq Polymerase
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Primers
Primers
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PCR Buffer
PCR Buffer
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Magnesium ions
Magnesium ions
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dNTPs
dNTPs
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Nuclease-free water
Nuclease-free water
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Denaturation
Denaturation
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Annealing
Annealing
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Extension
Extension
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Chelex Extraction
Chelex Extraction
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Positive control
Positive control
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Negative control
Negative control
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Gel Electrophoresis
Gel Electrophoresis
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Agarose
Agarose
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Ladder
Ladder
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Real-time PCR
Real-time PCR
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Reverse Transcriptase PCR
Reverse Transcriptase PCR
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Southern Blot
Southern Blot
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Flashcards
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Study Notes
Study Notes
Nucleic Acid Fundamentals
- Polymeric macromolecules are composed of repeating monomeric nucleotide units.
- There are two types: DNA and RNA.
DNA
- Polymer of deoxyribonucleotides storing and transferring genetic information.
RNA
- Polymer of ribonucleotides, synthesizes proteins and regulates gene expression.
Nucleotides
- Consists of three parts: sugar (pentose), nitrogenous base, and a phosphate group.
- The pentose sugar can be D-ribose or 2-deoxy-D-ribose.
Nitrogenous Bases
- Each chain monomer carries a heterocyclic base, or nucleobase, that is always linked to the 1' carbon if the sugar.
- Two types exist: purines and pyrimidines.
Bases Definitions
- Bronsted-Lowry definition: any molecule or ion that can accept a proton (H+). -ammonia (NH3) acts as a base by accepting a proton (H+) from water, forming ammonium ions (NH4+).
- Lewis definition: any molecule or ion that can donate a pair of electrons. -water (H2O) is an example, where each hydrogen atom donates one electron to the oxygen atom, forming two single bonds acts as a base.
- Arrhenius definition: a compound that produces hydroxide ions (OH-) when dissolved in water. -This definition is limited to aqueous solutions and primarily applies to ionic compounds that dissociate into hydroxide ions. -Sodium hydroxide (NaOH) releases hydroxide ions in water making it a base.
Key Characteristics of Bases
- Reacts with acids, producing salts and water (Neutralization)
- pH register above 7
- Many exhibit a slippery or soapy texture due to their protein interaction.
- Turns red litmus paper blue.
- Often taste bitter.
- Strong bases are corrosive to tissue
- Bases do not react with metals, unlike acids.
Phosphate
- Derived from phosphoric acid (H3PO4).
- It forms a phosphodiester link between two sugar residues.
- Functions as a strong acid.
Nucleoside Formation
- Nucleosides consist of a sugar + nitrogenous base.
- It's a two-subunit molecule where a pentose sugar is bonded to a nitrogen-containing heterocyclic base.
- Adenine + ribose results in adenosine.
- Adenine + deoxyribose results in deoxyadenosine.
Nucleotide Formation
- A nucleoside + phosphoric acid form this compound.
- PO4 forms a phosphoester bond with an -OH of the sugar, commonly at the 3'-OH or 5'-OH position.
- They are commonly named via the parent nucleoside with a "monophosphate" suffix.
- These compounds drive all metabolic processes thanks to being energy rich compounds
DNA Characteristics
- Adenine (A) bonds with thymine (T), and cytosine (C) bonds with guanine (G).
- Two strands are connected by hydrogen bonds between their bases.
- The double helix strands run antiparallel to each other or in opposite directions. -One strand runs in the 5'-to-3' direction, and the other is oriented in the 3'-to-5' direction
Central Dogma of Molecular Biology
- Prokaryotic ribosomes are composed of 70S with 50S and 30S subunits.
- Eukaryotic ribosomes are composed of 80S with 60S and 40S subunits.
Nucleic Acid Isolation Fundamentals
- Isolation of nucleic acid is a key procedure separating cells from debris, tissues, proteins, carbohydrates etc
- Stages of this procedure are: -Cell lysis happens with a mechanical or chemical method -Extraction is the separation of nucleic acids from the cellular components -Purification is achieved by DNA being precipitated by alcohol.
Specimen Collection
- Whole blood is the most common specimen for molecular diagnostics
- Other specimens taken are tissue, buccal swabs and bacteria/fungi
- Urine, feces, sputum, CSF, genital swabs, semen, etc. are also taken
- Formalin-fixed paraffin-embedded tissue, bone, hair, and nails are taken as forensic samples.
RNA Isolation Precautions
- Use RNase-free tubes, pipet tips, and reagents.
- Gloves should always be worn and work should be performed in a hood (PPE).
- It is best practice to use disposable items
- Treat liquids with DEPC (Rnase inhibitors during cell lysis like guanidine isothiocyanate).
- Cells or tissue must be rapidly and efficiently disrupted to preserve viability
- RNA must be partitioned selectively from DNA and protein.
- Store specimens in liquid nitrogen or at -80 degrees C, -20 degrees C, or long term at -70 degrees
- Avoid reusable glassware or bake for 4-6 hours at >270 degrees C (400 degrees C) to inactivate Rnases.
Extraction Methods: Liquid Phase: Organic Method
- Usage of Phenol and Chloroforr
- Acts as the "gold standard" of extraction methods.
- Yield high-quality DNA thanks to using a large mass of the sample for extraction
Extraction Methods: Liquid Phase: Inorganic Method
- Involves the incubation of nuclei with only proteinase K at 65°C.
- Incubation happens for more than 2 hours with Tris, EDTA, and proteinase K in a low salt buffer
- Extracted DNA can be used directly for diagnostic analysis -No additional purification required
- Saturated NaCl will precipitate protein.
- DNA is purified from the supernatant by 1-2ml of -20°C chilled absolute ethanol.
Extraction Methods: Solid Phase
- Offers increased ease of use and a high degree of reproducibility.
- Method will yield smaller amounts of DNA.
- This is most commonly used clinical DNA isolation method
- The basic method has usage of Minicolumn Purification
Extraction Methods: Solid Phase: Magnetic Beads
- A simple and efficient method.
- Magnetic carriers possess immobilized affinity ligands, or are prepared from biopolymer with affinity to target nucleic acids.
- Beads support the process thanks to larger binding capacity
Extraction Methods: Chelex
- Chelex works as a cation-chelating resin used in forensic applications.
- Chelex will be mixed with the sample specimen then boiled.
- Contaminating materials will bind to the resin which is removed by centrifugation.
- The DNA collected will be the supernatant.
Extraction Materials
- RNAse (ThermoFisher) degrades single stranded RNA
- Buffer 1 dissolves RNAse
- Lysozyme (Sigma) lyses Gram-negative bacterial cell wall
- Achromopeptidase(Sigma) lyse Gram-positive bacterial cell wall
- Sodium dodecyl sulfate (SDS) solubilizes cell membrane lipids
- Proteinase K (ThermoFisher) digests proteins
- Phenol: Chloroform: Isoanyl Alcohol separates DNA from other cellular materials
- Ethanol precipitates DNA from solution
- Tris-EDTA dissolves precipitated and dried DNA
Spectrophotometry Overview
- Optical instrument measuring light intensity at different wavelengths to calculate solution concentration.
- The absorption of the unknown is measured and compared to a reference standard.
- Maximum absorption for both DNA and RNA read at 260 nm (OD260) for the following solutions: -A 50 µg/mL solution of dsDNA -A 33 µg/mL solution of oligonucleotide -A 40 µg/mL solution of RNA
- dsDNA concentration can be calculated by: 50 µg/mL x A260 x dilution factor
Spectrophotometry - Purity of DNA
- The purity of DNA is measured with the A260/A280 ratio -If the ratio is 1.8, the sample is pure DNA. -If it is 2.0, the solution is pure RNA -A reading below 1.8 indicates contaminants like proteins, solvents -A reading above 2.0 indicated chloroform or phenol contamination -For the A260/A230 ratio, reading exceeding 1.4 is acceptable but under will indicated salt contamination
Nucleic Acid Amplification Types
- Target amplification: -Known nucleic acid sequence of interest is targeted for enzymatic replication. -Has usage of Polymerase Chain Reaction, Transcription-Mediated Amplification, and Loop-Mediated Amplification
- Signal amplification: -Method of detecting DNA target without direct template DNA amplification. -Relies on detection of substrates bound to the target nucleic acid. -Has usage of Branched DNA amplification
- Probe Amplification has reliance on the template DNA strand. -Ligase Chain Reaction and Strand Displacement Amplification are uses as part of reaction
Polymerase Chain Reaction (PCR)
- PCR was developed by Kary Mullis in the mid-1980s.
- This molecular technique involves in vitro enzymatic replication of defined DNA sequences, and functions as a DNA "copy machine”.
- PCR is one of the most useful techniques for speed and sensitivity in laboratories.
- First successful amplification utilized a short fragment of E.coli plasmid, pBR322
- First clinical amplification was amplification of beta globin for the diagnosis of sickle cell anemia.
Aspects of PCR
- It relies on amplification
- Requires temperature cycles.
- Taq polymerase is critical
- Requires key primers
PCR Key Steps
- Denaturation starts the PCR process
- Annealing for the key primer process
- Extension for lengthening the strands
- Repitition to keep making copies
PCR Components
- Template DNA, primers and nucleotides are needed
- Taq polymerase and reaction buffer are necessary
- Also needed is MgCl2
Template DNA
- It's best to utilize high-quality, purified DNA
- DNA extraction contaminants must be avoided
- A PCR replication template is of any DNA source including genomic DNA (gDNA), complementary DNA (cDNA), and plasmid DNA
Primers, Important points
- Reverse complements that define the region of the DNA that gets amplified.
- Carries free 3’OH groups
- A single end of forward and reverse primers hybridizes itself to the 3' end of each DNA strand
- Primers are specific keys that determine the success of the reaction
Primers Dos and Donts
- Do use 20-30 nucleotides in length
- Do use melting temperature (Tm)should between 55-65 C
- Do ensure the final Tm for the reverse and forward primers only differ by 2-3C
- Do ensure Optimal Gc content ranges between 40-60%
- Do ensure the 3'end of the primers would contain G or C to properly clamp primer
- Do not use secondary structures (hairpin) within each primer
- Do not use Dinucleotide repeats (AGAGAGAGAGA) or single base runs (GGGGG).
PCR Buffer
- PCR buffers provides a suitable chemical environment for peak activity and overall stability of the DNA polymerase.
PCR - Magnesium Ions
- MgCl2 will act as a cofactor for TaqPol
- Is a stabilizing agent for annealing, to reduce repulsion of template and primer
- Best practiced at 1.5-2.0mM
- Levels depend on template, buffer, DNA and dNTPs -Little Mg stops reaction, too much results in undesired products
PCR - DNTPs
- Building blocks of DNA
- Most often seen at 200 µM
- At (50-100 μM) enhances both the specificity and fidelity of the reaction , but reduces yield
- Yields particularly in long PCR are raised by high concentrations -Too high levels will harm fidelity and reduce PCR's effectiviness
DNA Polymerase
- DNA reaction depends on essential replication enzymes
- DNA must be used to make new strands
- To little polymerase stops process, too much harms specificity.
Nuclease-free Water
- Nuclease contaminants damage all components
- Free water must be stored in small aliquotes
- That way contamination will be contained
PCR - Temperature
- Initial denaturation at 95°C for 2 minutes is recommended -Avoid longer or higher temperature incubations
- Short (15-30 second) denaturation at 95°C should be often utilized
- In PCR, success depends on high ratio of specific to nonspecific annealing with the molecules
- Typical annealing temperatures are 5°C below the lowest primer's Tm. Often fall in the 50-60°C range -If too low non-specific, to hight stops reaction
- Typical annealing times are 15-30 seconds (Tm=(4 x(G + C)) + (2 X (A + T)°C)
PCR - Time
- As a general rule, time should be one minute per 1000 base pairs -3 minutes for a 3 kb product
- For products shorter than 1 kb, a reaction for 45-60 is effective
- Products more the 3kb require and more cycles require more time
Ways to Prevent Contamination
- Contamination is often stopped by proper space and time preparation
- Equipping the laboratory proper is important
- Seperatr pre and post reaction process
- Being careful about the environmental condition
- Regeants must be well sterilized
- Basic contamination will help the process
PCR Controls
- Positive controls must have sequence content to ensure sensitivity -Verifies the reaction and process are working
- Negative and blank tools must remove content to amplicons -Verfies no contamination exists
- Template should also be negative and remove sequence context -Verfies primer do not link non-target regions
Gel Electrophoresis
- Agarose concentration A linear DNA fragment of a given size migrates at different rates through gels containing different concentrations of agarose
Factors that determine the rate of migration of a DNA molecule
- Size of the molecule: faster/farther for smaller molecules A molecule's size is expressed as it's molecular weight - DNA conformation: supercoiled strand fastest. linear double standrd in the middle and circular the slowest
- Voltage applied: propotional to the molecule speed
- Rate of migration is proportional to the voltage applied
- increasing Voltage increases the molecule rate
-
Electrophoresis Buffer -Composition and ionic strength affects DNA mobility -If water electrical conductivity is minimal, DNA migrates slowly -High ionic strength (10X Buffer), the electrical conductance is efficient
-
Presence of DNA stains in the gel electrophoresis buffer -Addition to the gel may retard the rate of migration of the DNA
Materials needed for Agarose
- The major chamber it will run through
- Proper Agarose material which can be suspended in a drying try
- Buffer for storage
- Staining agent for readability
- Combe for well forming
- DNA Ladder to track changes
- Sample of the tested substance
- and the correct power sypply
Gel - Stain
- The key compound can adhere and be stimulated with UV
- Commonly used for DNA tracking within the wells
- Best practices are .5 -1 of use
Gel: Ladder
- Well known ladder creates markers to measure
- These come in pre-set sizing scales
- These scales help quickly estimate base pair size
Gel - Loading Dye
- Mix of gycol and tracking dyes
- Load weight on sample through density
- Dye is easy to see to watch tracking for the machine
Gel- UV Light
- Allows for flourenset tracking to be easy to visually assess
- This has quantification for the sample
Common Gel Question
- When selecting the 5% va 2%, agarose should be considered
- Common load buffers contains multiple reagents -gycol -tracking markers -ions
- Methylene or crystal violet are common tracking tools
PCR Variant - Real TIme
- Real Time (RT) PCR monitors on what happening with the DNA inside
- This has non-secific duets that are double stranded -Can utilize unique chains of oligionulceotides
Flow of Sample and RT-PCR
- Take Sample and place it in swab
- Extract and copy into a Viral Extracted RNA
- Enter the real time and electrophoresis -With this the curves an be made
Signal Strength
- Cycle treshold is the change for an area
- RT can be flourescent, FRET -Some have 3 chain connections from primer, qeuncer, and RT-Poly chains
Real Time Assays
- A series of reverse process and poly chaning for PCR can be combined for testing
- Qualitative results can be achieved
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