Gen 3040 final
How many mL of 100% ethanol are needed to prepare 200 mL of a 75% v/v solution of ethanol?
150 mL
Determine the molarity of 12.2 g of sodium chloride (MW:58.44 g/mol) dissolved in 75.0 mL of water.
2.78 M
For all samples collected during this week's purification, you will need to determine a sample's relative purity by comparing the ratio of a sample's absorbance at __________nm OVER a sample's absorbance at ___________nm.
260 nm/ 280 nm
How many amino acids make up the chromophore in GFP?
3
If a circular piece of DNA has three sites for a particular restriction enzyme, into how many fragments will that restriction enzyme cut the DNA?
3
A popular enzyme tag (or label) used in ELISA is the enzyme HRP. Many ELISA methods (including today's lab) will make use of what substrate molecule that is subsequently oxidized by HRP?
3,3',5,5' tetramethylbenzidine (TMB)
You are making your standards for analysis by the BCA assay using a standard stock of 2 mg/mL of BSA protein. To make a total of 1000 µL standard for analysis with a final [BSA] concentration of 100.0 µg/mL, what volume (in µL) of the standard stock solution will you need to add to your Bradford assay?
50
At which of the following melting temperature (Tm) should you design your primer?
78 degrees Celsius
In the context of gene cloning, what is a vector?
A plasmid used to house a gene of interest in order to transmit it from one place to another.
What is recombinant DNA (rDNA)? Select the best answer.
A sequence constructed of DNA from more than one organism.
For lab 10, you will determine which of the following? Please select any that may apply!
Activity and specific activity of alcohol dehydrogenase for EtOH, The effect of pH on the activity of alcohol dehydrogenase, The effect of temperature on the activity of alcohol dehydrogenase
From which organism was green fluorescent protein (GFP) first isolated?
Aequorea victoria
What is the name of the enzyme are we using for our lab 10?
Alcohol Dehydrogenase
Why would you add a restriction site via a silent mutation in your primer?
Allows you to verify successful mutation of the gene through electrophoresis
Of the three assays discussed in detail for this week, the __________ assay utilizes the colorimetric compound bicinchoninic acid (abbreviation ok).
BCA bicinchoninic acid
Which feature lends GFP its glowing property?
Chromophore
Which method will be applied in the current week's lab using SnapGene to verify the desired pieces of DNA are of the correct size?
DNA Agarose Gel Electrophoresis
What enzyme do we use to "join" or paste our desired gene and target vector together?
DNA ligase
Which of the following correctly lists the order of the steps in PCR?
Denaturation, annealing, elongation
Data obtained from various methods using absorption spectroscopy can be helpful in determining all of the following EXCEPT: Determining the exact order of the amino acid sequence in a protein Determining sample purity. Determining a protein's secondary structure Determining different chemical bonds in a protein Determining certain types of molecular interactions
Determining the exact order of the amino acid sequence in a protein
In a brief-but-detail paragraph using complete sentences, please compare and contrast the 3 different formats (or types) of ELISA methods discussed in the Lab 12 handout.
Direct ELISA is the fastest and simplest; though the cost is higher. Direct ELISA has a lower level of sensitivity. Like direct ELISA, indirect ELISA has the same "direct capture" method; however, it requires extra steps, has higher sensitivity, and is more cost effective. In indirect, Sandwich ELISA, the antigen does not bind directly to the plate and the capture method is specialized. Sandwich uses direct and indirect methods based on labelling.
What does ELISA stand for?
Enzyme Linked Immunosorbent Assay
From the options below, assign the correct, overall "sequence" for the essential steps for today's affinity-based purification of a GST-tagged protein.
Equilibration, binding, wash, elution
All of this week's assays can be considered "non-destructive", which implies that a sample can be re-used after treatment and analysis.
False
True or False: The Bradford standard curve is created by performing a serial dilution using a sample of an unknown concentration in order to generate a best-fit (or "linear regression") line that can be used to determine the concentration of another unknown sample.
False
True or False: In this week's Labster simulation, adding doxycycline to the media will upregulate the expression of the RAD52 gene, which is important for DNA repair.
False - it will silence gene expression
True or False: One general property of fluorescent proteins is their stability, and any changes to their local environment will not produce any variations.
False, changes to the local chromophore environment will also produce dramatic variations in the fluorescent protein's spectral characteristics.
Why is it important to control the G-C base pair percentages in a primer?
G-C base pairs having 3 hydrogen bonds affects adherence and melting temperature
In the Molecular Cloning Labster simulation, which gene is used as a reporter gene to monitor the expression level of the target gene?
GFP
Why is GFP useful in many molecular biology experiments?
GFP can track localization of proteins, GFP can be used to track gene expression, GFP can be mutated to different colored derivative for multiple tracking
What protein is being purified in this week's lab? PLEASE BE SPECIFIC and MIND YOUR SPELLING!!! Either the full name or the abbreviation will be sufficient!
GST::EGFP
The elution buffer utilized in lab this week contains a "specific" compound that releases any bound GST::EGFP recombinant protein from the immobilized resin in the column. What is this compound?
Glutathione
What does "GST" stand for? Please provide the FULL name, no abbreviations. Don't forget to mind your spelling!
Glutathione S-transferase
After the lysis of a cell culture, the large, insoluble cellular components (i.e., membranes) can be removed from a raw cell extract through which of the following methods below?
High-speed centrifugation
Imagine you're working in a research lab, and you endeavoring to design an antibody that will bind to a single epitope with minimal non-specific binding. Should your design be based on a monoclonal or polyclonal basis and WHY?
If the desire is to produce an antibody with specific binding to a single epitope, a monoclonal basis is best suited. Monoclonal mixtures, by definition, are designed with this purpose.
Comparing the BCA and Bradford assays for protein quantification, a major disadvantage of the BCA assay is (please select any that may apply):
It is more time consuming.
The pET-TIGER recombinant plasmid contains several important proteins with specific functions. Which of these proteins allowed for selective growth using the antibiotic kanamycin?
KanR
What enzyme is responsible for joining two fragments of DNA to generate recombinant DNA?
Ligase
"Fixing" is an important step prior to staining gels. Older, original methods used a "fixing solution" that usually included methanol and acetic acid. What is the importance of the methanol in these types of solutions?
Methanol extracts SDS from the gel so that SDS does not interfere with staining.
What are the benefits of genetic engineering?
Modification of plants for pest and disease resistance, Treatment of diseases, Producing food with desirable traits
ADH incorporates the use of the redox pair [ ___________________________] as part of its enzymatic reaction. A byproduct of its reaction, NADH , is responsible for a colorimetric change that can be measured at [________] nm.
NAD+/NADH and NADH 340 nm
Which of the following online tools will be used in the lab for this week?
NEB Tm Calculator, SnapGene, WatCut
Which of the following do modern plasmids contain?
Origin of Replication, Multiple Cloning Site, Selectable Marker
Which of the following lists the steps of vector assembly in the correct order?
Plasmid isolation, digestion, ligation
What does "PAGE" stand for (three words); please use the full name, no abbreviations.
Polyacrylamide Gel Electrophoresis
Which program will be used to look at/visualize the structure of GFP in this week's activity?
PyMOL
To investigate sequence similarities, you can align ___ sequences to identify conserved regions.
RNA, DNA
Which one of the following best describes restriction enzymes?
They are used as a powerful tool to manipulate DNA in molecular biology, They are part of a bacterium's defense mechanism, They recognize and cleave a specific nucleotide sequence
How has green fluorescent protein (GFP) been mutated for molecular biology applications?
To increase maturation rate and folding rate, To create different colored mutants, To enhance protein stability
Where will bands with larger DNA fragments appear on an agarose gel? Read all of the answer choices carefully and select the best answer.
Towards the top of the gel (above bands with smaller DNA fragments).
True or False: Multiple digestions can occur in a single buffer.
True
True or False: The restriction enzyme EcoRI recognizes a specific point known as the recognition site on the DNA and leaves a sticky end.
True
True or False: The use of a reporter construct such as Green Fluorescent Protein (GFP) is a common technique to examine levels of gene expression.
True
True or False: Using BLAST, sequence alignments can be performed given a query DNA sequence and compared to matches from a database. Results or "hits" with lower expectation values (E-values) share higher similarities with the query DNA sequence.
True
True or False: When using SnapGene, there is a feature annotated as "Fusion Protein" in the Map view of a pET28a plasmid. This is the protein the plasmid (or expression vector) is designed to express in bacteria.
True
True or false: The registration code for SnapGene provided to students for this course is good for only one personal computer.
True, it is not transferable so choose the appropriate device carefully.
True or False: There can be multiple cut sites for a single restriction enzyme in one plasmid.
True, one restriction enzyme can have multiple cut sites in a plasmid
Considerations you should take into account when deciding on a targeted mutation for this week's lab include which of the following?
What type of mutation will occur, What the literature says (scientific publications), What you are trying to achieve
The region/location within the enzyme where the substrate binds is called the:
active site
In order to calculate the specific activity of a given enzyme, we must first calculate the enzyme's ________________ under defined conditions. We also need to know the relative____________ of enzyme in the sample.
activity, concentration
For this lab, which "type/method" of column chromatography will be be using to isolate and purify our GST:EGFP fusion protein?
affinity
The ELISA method incorporates a molecule known as an "antigen", with which a specifically selected antibody will interact. In nature, an antigen is:
any molecule that induces an immune response.
For the purposes of analysis of the SDS-PAGE gel, you will need to calculate the "Rf" value (or the "retention factor/value") for several, specified bands on your gel. This value is calculated as " __________" divided by "__________ ".
band travel distance BY total length of the gel
Which of the following components found in the SDS Loading Buffer are used to (or capable of) denature proteins for analysis by SDS-PAGE? Please select all that may apply!
beta-mercaptoethanol, sodium dodecyl sulfate (SDS)
When planning to purify or isolate a target protein, you should consider each of the following physical/chemical properties of proteins EXCEPT: isoelectric point (charge) color hydrophobicity estimated size
color
Which fluorescent protein was developed by mutating serine at position 65 to a threonine residue and has characteristics/advantages including it can be imaged using commonly available filter sets and it is very bright?
enhanced green fluorescent protein (eGFP)
Concerning the change in absorbance for a reactant or product over time, the observation of a "plateau" region suggests the reaction has reached _______________.
equilibrium
PCR amplifies a DNA fragment _____.
exponentially
For the purpose of ELISA, a "reporter" enzyme or tag is always tagged (or "labeled) on a secondary antibody.
false
It is the specialized, unique "epitope" region of every antibody that subsequently binds to a unique "paratope" region of a specific antigen.
false
TRUE or FALSE: For the purposes of last-weeks purification...we utilized a GST-tag that is attached to the c-terminus "end" of the EGFP protein.
false
TRUE or FALSE: In similar fashion to DNA gel electrophoresis, samples containing proteins are denatured/treated and given an overall positive charge. During the SDS-PAGE run, this allows the proteins to migrate towards the negative electrode of the gel.
false
True or False: ELISA can only be used for analysis of proteins and/or polypeptides.
false
True or False: Enzymes act as a catalyst by increasing the activation energy of the reactions, therefore speeding up the reactions and decreasing the free energy exchange of the reaction.
false
Individually speaking...the most important factor that affects an enzyme's activity is typically the __________ of the enzyme's local environment.
pH
Prior to running your SDS-PAGE gel, you will need to enzymatically "cleave" the GST-tag from your recombinant fusion protein (purified in Lab #8). The enzyme utilized for this is _______________.
thrombin
For a successful PCR, you must ensure that the polymerase has been included in the reaction mix at the proper concentration. The role of polymerase in a PCR reaction is which of the following?
to catalyze the synthesis of DNA or RNA polymers whose sequence is complementary to the original template strand
The "peak" position on an free energy diagram following a reaction's progress from reactants/substrates to the products is called the "____________________"
transition state
From a structural perspective, the majority of an antibody's final structure is highly conserved, with the exception of a pair of variable regions on the "arms" of the antibody.
true
Generally speaking, for most enzymes, an increase in temperature will often correlate to an increase in activity (until an optimum point is reached).
true
TRUE or FALSE: Larger proteins (>50 kDa) will migrate better on lower-percentage gels (< 8-10%), while higher percentage gels (> 12-15%) can be used for smaller proteins (< 50 kDa).
true
TRUE or FALSE: Proteins can be characterized as "amphoteric", meaning they can maintain both positive and/or negative charges in the same molecule under various conditions.
true
True or False: Absorbance is a unit-less measurement.
true
True or False: For the purpose of lysing cells, a detergent's mode of action (such as SDS or B-PER) is to degrade and/or solubilize a lipid bilayer.
true
True or False: The extinction coefficient for a given compound is constant, so long as the assay parameters (wavelength, temperature, solvent, etc.) are also held constant.
true
True or False: The restriction digest uses restriction enzymes to prepare both the vector plasmid and the gene insertion.
true
True or False: We should expect the cytosolic GST::EGFP recombinant protein to be soluble.
true
The UV280 assays operates based on the varying presence of the which amino acid(s) in the polypeptide sequence of a given protein. Please select any that may apply!
tryptophan, phenylalanine, tyrosine
Both the BCA and Bradford assays incorporate the use of a compound that change color in the presence of a protein that is visible to our eyes; therefore, these two compounds absorb light in the __________ range.
visible
