BIOLOGY 111 EVALUATION EXAM QUESTIONS AND
ANSWERS GRADED A+
✔✔What organism was Taq polymerase isolated from? - ✔✔Thermus aquaticus:
thermophilic bacterium (heat tolerant) that live in hot springs
✔✔What is multiplex PCR? - ✔✔Amplification of multiple targets into a single PCR
experiment by utilizing multiple primer pairs in a single reaction mixture
✔✔What is the purpose of gel electrophoresis? - ✔✔Allows you to separate a mixture of
different sized DNA molecules so that DNA samples can be compared to other known
DNA profiles.
✔✔What is the charge of DNA? - ✔✔DNA has a negative charge
✔✔What direction should DNA run? - ✔✔Toward the positively charged end (anode)
✔✔What are things that influence how far DNA will run? - ✔✔Charge of molecule: DNA
carries a negative charge so attracted to positive end • Size: Smaller
molecules/fragments will travel faster and further • Density of gel: the more dense the
gel is, the slower the particles will move. You want a gel that is dense enough to
separate particles but that won't take too long
✔✔Who will run further from a well? 700bp of DNA or 200bp of DNA. - ✔✔200 bp
(smaller, so travels faster and further)
✔✔Why is developing a DNA profile important? - ✔✔DNA testing allows forensic
investigators to identify a guilty individual with a high degree of certainty because the
DNA sequence of every person is unique (except for identical twins)
✔✔How did multiplex PCR help in our analysis versus just using a single pair of
primers? - ✔✔A single pair would not allow you to distinguish between
samples/individuals with a high degree of certainty
✔✔Why are DNA ladders (standards) important? - ✔✔They are used to determine the
sizes of bands in other DNA samples. Thus, we are able to compare them.
✔✔Can you tell the difference between a homozygote or heterozygote gene on a gel? -
✔✔Yes, a homozygote will have one band at a particular loci and a heterozygote will
have two bands at a particular loci
✔✔What was the purpose of using SYBR Green in the gels? - ✔✔SYBR Green binds to
DNA and fluoresces when exposed to the right wavelength of light. This makes the
separated bands visible.
, ✔✔What was the purpose of pouring the buffer in the electrophoresis chamber? -
✔✔Buffer controls the net charge of molecules by maintaining the pH at nearly neutral.
This facilitates proper migration and separation of DNA molecules.
✔✔What is the central dogma of gene expression? Define transcription and translation.
- ✔✔The central dogma states that genes specify the sequence of mRNAs, which in
turn specify the sequence of amino acids making up all proteins o Transcription: DNA
from a gene sequence is copied to mRNA o Translation: directs the synthesis of
proteins
✔✔What is the name of the primary enzyme used in this lab? Where is it coming from?
- ✔✔B-galactosidase is an enzyme is E. coli that is encoded by the LacZ gene when E.
coli needs to break down lactose
✔✔What are the components of the lac operon and what do they do? - ✔✔Operon: is a
functioning unit of DNA containing a cluster of genes under the o RNA polymerase:
needed to start transcription and produce mRNA o Promoter: the binding site for RNA
polymerase o Operator: negative regulatory site that the repressor can bind to o CAP
site: positive regulatory site that catabolite activator protein (CAP) can bind to o
Repressor: when the repressor is bound it blocks RNA polymerase and prevents the
production of mRNA (so no production of proteins) o The lac operon has 3 genes that
help the cell utilize lactose: lacZ - encodes β-galactosidase enzyme, which splits lactose
into monosaccharides. lacY - encodes lactose permease protein, which is a
transmembrane "pump" that allows the cell to import lactose. lacA - encodes
transacetylase enzyme
✔✔E. coli makes B-gal to breakdown what sugar? - ✔✔Lactose
✔✔Expression of the LacZ gene causes the production of what enzyme? - ✔✔B-gal
✔✔How can ONPG be used to measure optimal growth conditions for the expression of
LacZ? - ✔✔Because B-gal also breaks down ONPG and one of the products of this
reaction has a yellow color (ortho-nitrophenol), we can investigate the regulation of
LacZ transcription by observing the rate of color change in the tubes. Presence of
yellow color would mean that B-gal was produced, thus LacZ would be expressed.
✔✔How does LacZ/B-gal expression influence activity? If LacZ/B-gal expression is
high/low, how quickly do you think ONPG will be broken down into galactose and o-
nitrophenol? - ✔✔If LacZ/B-gal expression was high, ONPG would be broken down
quickly because there would be more enzymes available.
✔✔Understand how LacZ expression changes when E. coli is grown under different
culture conditions: LB (plain media), LB+lactose, LB+glucose, and LB+glucose+lactose.
ANSWERS GRADED A+
✔✔What organism was Taq polymerase isolated from? - ✔✔Thermus aquaticus:
thermophilic bacterium (heat tolerant) that live in hot springs
✔✔What is multiplex PCR? - ✔✔Amplification of multiple targets into a single PCR
experiment by utilizing multiple primer pairs in a single reaction mixture
✔✔What is the purpose of gel electrophoresis? - ✔✔Allows you to separate a mixture of
different sized DNA molecules so that DNA samples can be compared to other known
DNA profiles.
✔✔What is the charge of DNA? - ✔✔DNA has a negative charge
✔✔What direction should DNA run? - ✔✔Toward the positively charged end (anode)
✔✔What are things that influence how far DNA will run? - ✔✔Charge of molecule: DNA
carries a negative charge so attracted to positive end • Size: Smaller
molecules/fragments will travel faster and further • Density of gel: the more dense the
gel is, the slower the particles will move. You want a gel that is dense enough to
separate particles but that won't take too long
✔✔Who will run further from a well? 700bp of DNA or 200bp of DNA. - ✔✔200 bp
(smaller, so travels faster and further)
✔✔Why is developing a DNA profile important? - ✔✔DNA testing allows forensic
investigators to identify a guilty individual with a high degree of certainty because the
DNA sequence of every person is unique (except for identical twins)
✔✔How did multiplex PCR help in our analysis versus just using a single pair of
primers? - ✔✔A single pair would not allow you to distinguish between
samples/individuals with a high degree of certainty
✔✔Why are DNA ladders (standards) important? - ✔✔They are used to determine the
sizes of bands in other DNA samples. Thus, we are able to compare them.
✔✔Can you tell the difference between a homozygote or heterozygote gene on a gel? -
✔✔Yes, a homozygote will have one band at a particular loci and a heterozygote will
have two bands at a particular loci
✔✔What was the purpose of using SYBR Green in the gels? - ✔✔SYBR Green binds to
DNA and fluoresces when exposed to the right wavelength of light. This makes the
separated bands visible.
, ✔✔What was the purpose of pouring the buffer in the electrophoresis chamber? -
✔✔Buffer controls the net charge of molecules by maintaining the pH at nearly neutral.
This facilitates proper migration and separation of DNA molecules.
✔✔What is the central dogma of gene expression? Define transcription and translation.
- ✔✔The central dogma states that genes specify the sequence of mRNAs, which in
turn specify the sequence of amino acids making up all proteins o Transcription: DNA
from a gene sequence is copied to mRNA o Translation: directs the synthesis of
proteins
✔✔What is the name of the primary enzyme used in this lab? Where is it coming from?
- ✔✔B-galactosidase is an enzyme is E. coli that is encoded by the LacZ gene when E.
coli needs to break down lactose
✔✔What are the components of the lac operon and what do they do? - ✔✔Operon: is a
functioning unit of DNA containing a cluster of genes under the o RNA polymerase:
needed to start transcription and produce mRNA o Promoter: the binding site for RNA
polymerase o Operator: negative regulatory site that the repressor can bind to o CAP
site: positive regulatory site that catabolite activator protein (CAP) can bind to o
Repressor: when the repressor is bound it blocks RNA polymerase and prevents the
production of mRNA (so no production of proteins) o The lac operon has 3 genes that
help the cell utilize lactose: lacZ - encodes β-galactosidase enzyme, which splits lactose
into monosaccharides. lacY - encodes lactose permease protein, which is a
transmembrane "pump" that allows the cell to import lactose. lacA - encodes
transacetylase enzyme
✔✔E. coli makes B-gal to breakdown what sugar? - ✔✔Lactose
✔✔Expression of the LacZ gene causes the production of what enzyme? - ✔✔B-gal
✔✔How can ONPG be used to measure optimal growth conditions for the expression of
LacZ? - ✔✔Because B-gal also breaks down ONPG and one of the products of this
reaction has a yellow color (ortho-nitrophenol), we can investigate the regulation of
LacZ transcription by observing the rate of color change in the tubes. Presence of
yellow color would mean that B-gal was produced, thus LacZ would be expressed.
✔✔How does LacZ/B-gal expression influence activity? If LacZ/B-gal expression is
high/low, how quickly do you think ONPG will be broken down into galactose and o-
nitrophenol? - ✔✔If LacZ/B-gal expression was high, ONPG would be broken down
quickly because there would be more enzymes available.
✔✔Understand how LacZ expression changes when E. coli is grown under different
culture conditions: LB (plain media), LB+lactose, LB+glucose, and LB+glucose+lactose.
