Final Exam Review: ELISA, PCR, Bacterial Transformation, Spectrophotometry, Phosphatase Assay, Protein Separation

These flashcards cover key concepts and processes related to ELISA, PCR, bacterial transformation, spectrophotometry, acid phosphatase assays, and protein separation techniques, providing a comprehensive review for the final exam.

What is the basic process of an ELISA?

In ELISA, an antigen binds to a specific antibody, and a positive or negative result indicates the presence or absence of the target substance.

Define negative and positive controls in an experiment.

Negative controls are samples that should not produce a result, while positive controls are samples that are expected to produce a positive result, ensuring the test works correctly.

What is the purpose of including primers in a PCR reaction?

Primers are essential as they provide a starting point for DNA synthesis during PCR amplification.

What type of DNA can be used as a template in PCR?

Any form of DNA, including contaminated or impure samples, can be used as a template in PCR.

What are the three basic steps in a PCR cycle?

The three basic steps are denaturation, annealing, and extension.

Is PCR amplification linear or exponential?

PCR amplification is exponential.

What determines the size of the PCR product?

The design of the primers determines the size of the PCR product.

Why is MgCl2 included in the PCR reaction mix?

MgCl2 is a cofactor for the DNA polymerase enzyme, which is crucial for the reaction.

What is genetic transformation?

Genetic transformation is the process of introducing foreign DNA into a bacterial cell.

What are constitutive and inducible gene expression?

Constitutive expression refers to genes that are continuously expressed, while inducible expression refers to genes that are expressed only under certain conditions.

Which plates in the bacterial transformation experiment served as control plates?

The LB and LB/amp plates served as control plates, ensuring that the experiment's setup is valid.

What factors influence enzyme activity in biochemical reactions?

Factors such as temperature, pH, substrate concentration, and enzyme concentration can increase or decrease enzyme activity.

What is the purpose of the standard curve in spectrophotometry?

The standard curve is used to establish the relationship between absorbance and concentration for quantifying samples.

What is the reaction catalyzed by acid phosphatase?

The reaction involves the hydrolysis of phosphate esters to produce an alcohol and inorganic phosphate.

What defines the four levels of protein structure?

The four levels are primary (amino acid sequence), secondary (folding patterns), tertiary (3D shape), and quaternary (subunit interactions).

What determines the migration direction of proteins in gel electrophoresis?

The migration direction is determined by the charge of the proteins and the electric field applied to the gel.

Basic process of ELISA

1. add sample to wells,

proteins and antigens bind to the plastic via hydrophobic interactions

2. add primary antibody, which binds to the antigens

3. add enzyme labeled secondary antibody, which binds to the primary antibody

4. add substrate, which detects if the enzyme is present or not

Positive control

contains variable we are testing for; Confirms that your test or experiment runs correctly

Negative control

does NOT contain the variable we are testing for ; confirms experiment does not generate false positives

Positive well

contains antigen, primary/secondary antibodies, and substrate; our experiment- turned blue

Negative well

only contains substrate floating around

What could generate false positive?

Errors in washing out wells, cross contamination of antigen

What could generate a false negative?

Forgot to add a reagent

What is the purpose of a PCR reaction?

to amplify specific regions of DNA that can be used for size determination, restriction mapping, etc.

What are essential components of PCR reactions?

Target DNA

pair of primers

free deoxynucleotides

taq polymerase

buffer with cofactor Mg++

What kind of DNA can be used for template?

crude or pure

What are the properties of PCR primers?

1. need 2 primers usually 20 nucleotides in length

2. designed to flank the region to be amplified

3. must be complementary to 3' end of gene being amplified

4. provided in large excess

What is special about Taq polymerase?

comes from Thermus aquaticus bacteria in hot springs and can survive extremely hot temperatures (75˚ C)

What are the three basic steps in each cycle? (temp used and purpose of each step)

1. Denaturation- 95°C used to separate DNA into single strands

2. Annealing- 55°C used to anneal (binding) primers to target areas in template DNA strands

3. Extension- 72-75°C, Taq polymerase adds free deoxy nucleotides to primer

Is DNA amplification in PCR linear or exponential?

exponential

What determines size of a PCR product?

Location of primers (distance between them)

Two functions of dNTPs?

Nucleotides to grow strand and energy (from dATP)

What is purpose of MgCl2 in PCR?

cofactor in the buffer to improve efficiency and amplification

Function of Chelex in PCR?

Protects DNA by removing contaminating metal ions that catalyze DNAase reactions (should not be present in PCR reaction tube bc it will bind with Mg and cause reaction to fail)

Function of boiling in PCR?

releases DNA and inactivates cellular proteins that can interfere with reaction

How to find out if you have obtained expected PCR product?

Run DNA agarose gel electrophoresis

What is the basis of separation of DNA on an agarose gel?

Size of fragments

How is DNA electrophoresis different from PROTEIN electrophoresis?

Ethidium bromide staining gives fluorescence under UV light

Wells at end allow DNA to migrate from cathode to anode

in protein gel, wells are in the center and no ethidium bromide present

What gives DNA the negative charge?

phosphate group

What affects the migration rate of DNAs in agarose gel electrophoresis?

Size of fragment (bigger = slower rate), agarose concentration (higher = slower rate), buffer, voltage applied, conformation of DNA

What chemical stains DNA?

ethidium bromide, glows under UV light

Purpose of lane marker in gel?

estimate size of fragment

How does the size of linear DNA molecules relate to their migration rate?

Migration rate is inversely proportional to the log10 of their molecular weight

What two blue dyes are included in the marker? Why?

Bromophenol blue comigrates with 300bp fragments and Xylene cyanol comigrates with 9kb fragments (visual monitoring of migration)

What is gel electrophoresis?

A DNA fragment that has been cut with restriction enzymes can be separated using electric field

0.5% gel

fragments larger than 10kb

1% gel

500 bp-10 kb

2%

smaller than 1 kb

Functions of loading dye?

- Does not stain DNA

- Allows monitoring of movement of DNA fragments

- Makes loading samples easier (color and glycerol for density)

What is genetic transformation?

Process to introduce a specific gene into an organism and have that genetic information expressed by that organism

What are competent cells?

Cells are passively made permeable to plasmid DNA using lab procedures (calcium chloride and heat shock)

constitutive gene expression

gene is always expressed (beta lactamase and araC)

inducible gene expression

specific conditions needed to turn on gene (temp, chemicals, etc. in our experiment- arabinose turns on GFP gene)

Process of turning "on" GFP gene

Arabinose binds to araC, changes conformation of protein, promoter region of DNA is now exposed and available for transcription, RNA polymerase can now bind to promoter region

What all needs to be present for transcription of GFP mRNA in transformed E coli?

arabinose

GFP gene

promoter

araC protein

Important properties of E coli

single celled, reproduces quickly, doesn't infect animals or plants, not naturally antibiotic resistant

Important properties of pGLO

selectable marker = ampicillin resistance gene which codes for beta lactamase protein, origin of replication, "polylinker" area, GFP gene, PBAD promoter and araC gene (special gene regulation system for GFP)

How is bacterial transformation set up in this experiment?

1. Host cells E Coli

2. Plasmid contains selectable marker and gene of interest (beta lactamase and GFP)

3. incubation, heat shock, recovery allows for plasmid DNA into host

4. Selection mechanism- growth on media containing antibiotics

Why do we use LB/amp media to select for transformants?

Only transformed bacteria have ampicillin resistance gene and grow on amp plates

What was the purpose of the arabinose?

Turn on GFP gene

Which plate---LB/amp or LB/amp/arab--- produced GLOWING colonies?

Amp/ara because it contained arabinose to turn on GFP gene, allowing for GFP to be produced and glow

What are the control plates? What are their purposes?

-pGLO plates: factor being tested not applied (LB- make sure bacteria were alive/growing, LB/amp- shows untransformed bacteria do not have amp resistance gene)

Parallel dilution

C1V1 = C2V2

What is an absorption spectrum?

a graph plotting a pigment's light absorption (of known concentration) versus wavelength, where we can find λmax

Relationship between absorbance and concentration

linear

What is λmax?

The wavelength where the maximum absorbance occurs, determined by finding wavelength with highest absorption on absorption spectrum

Purpose of the standard curve?

Relates absorbance to concentrations based on Beer's law, can be used to extrapolate concentration of unknown solution

Reasons we measure curve at λmax?

Sensitivity and accuracy

What is Beer's Law and how does it relate to standard curve?

Beer's Law: the concentration of a chemical solution is directly proportional to its absorption of light

A= bεc, A: Absorbance, b: pathlength (cm), c: molar concentration (mol/L), ε: molar absorptivity coefficient (L mol-1 cm-1)

y = absorbance, x = concentration (c), m = ε

using absorbance we can find concentration - *** if in mM, convert to M (1 mM = 0.001 M)

Standard curve

Absorption v concentration

What influences activity of enzyme?

pH, temperature, amount of enzyme (more enzyme increases reaction rate), substrate concentration, effect of activators/inhibitors