Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

biochemical methods > exam 2 > Flashcards

exam 2 Flashcards

1
Q

__________ relaxes supercoiled DNA

A

DNA gyrase (function)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

__________ breaks hydrogen bonds to make DNA single-stranded

A

DNA helicase (function)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

DNA Polymerase removes errors by a __________ activity

A

3’-5’ exonuclease (what uses this)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Isopropanol allows for __________; Addition of __________ can also aid in this

A

DNA precipitation; salt (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

The FWD primer anneals to the __________, and is a small part of the same sequence as the __________.

A

3’ end of the compliment strand; 5’ end of the coding strand (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

The REV primer anneals to the __________, and is a small part of the same sequence as the __________.

A

3’ end of the coding strand; 5’ end of the complement strand (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Ideal primer length is __________

A

20-30BP (what is this range for)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

In PCR, the product which has the desired gene region appears only in the _________

A

third cycle (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

The reverse primer is the reverse complement of the __________

A

end of the coding strand (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What direction does DNA synthesis happen?

A

5’ to 3’ (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Good primer Tm is between __________

A

52-65C (what is this range for)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the Tm of a primer?

A

The point at which half of the DNA is single-stranded (what is this describing)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How much GC content should a primer have?

A

50-60%, with more at the 3’ end (GC clamp) (what is this rage for)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to __________

A

LOG(MW) (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Plasmids can replicate __________

A

independently (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

__________ are modified plasmids used in recombinant DNA technology for cloning.

A

Vectors (define)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

A reporter gene is a __________ gene

A

drug resistance (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

The __________ is where the gene can be inserted.

A

MCS (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

In TOPO cloning there is a __________ in one of the strands of the vector. The PCR insert will have a matching __________ at the 5’-end of the PCR product.

A

GTGG overhang; CACC region (what are these sequences related to)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

pET101D has T7 promotor and terminator, these are essential for __________ to transcribe the gene.

A

T7 polymerase (what does this do in pET101D)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

LacO is an Operator/DNA (regulatory) sequence that controls the __________ in TOPO cloning

A

protein expression (what controls this in TOPO cloning)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

LacI protein binds to LacO and prevents activity of __________.

A

T7 RNA polymerase (what prevents this)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Upon induction with a lactose analog, IPTG, LacI binds to IPTG and leaves the operator (LacO) free, permitting __________

A

protein synthesis (what binds LacI to induce this)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

TOPO cloning includes a V5 epitope and c-term his-tag, if you do not include the __________

A

STOP codon (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

What are these advantages of TOPO Cloning

A
  • Doesn’t require restriction digest
  • Directional insertion
  • No ligation required
  • Relatively time saving
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What are these disadvantages of TOPO Cloning

A
  • Overhangs sensitive to degradation
  • Must purchase a vector each time
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

TA cloning uses a Taq polymerase that adds __________ to the __________ of the PCR product.

A

A; 3’ end (what adds A to the 3’ end of PCR product)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What are these steps for Plasmid Purification

A
  • Grow cells in abx, then pellet
  • Resuspend
  • Lyse
  • Neutralize pH
  • Capture plasmid DNA, remove excess salt
  • Elute
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Number of double stranded DNA copies that span the size between the primers after n cycles of PCR:

A

(2^n - (n*2)) (what does this eqn represent)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

__________ are endonucleases commonly found in bacteria or archaea and they provide defense mechanism against foreign DNA

A

Restriction Enzymes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

Restriction enzymes have specific recognition sequences and cleavage site, and they cut DNA sequences in a __________ manner

A

predictable (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

The restriction enzyme recognition sequences are __________

A

self-complementary (what sequences are self-complementary)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

RNA and DNA sequences that code for protein are called __________

A

open reading frames (ORF) (define)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

Each RNA/DNA strand has __________ ORF’s

A

3 (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

What are these advantages of TA Cloning

A
  • Doesn’t require restriction enzymes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

What are these disadvantages of TA Cloning

A
  • Vector and insert sensitive to degradation
  • Non-directional cloning
  • Taq-polymerase has low proofreading ability
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

For deciding the vector for pET28 cloning, choose two different __________ that are __________ within your gene of interest

A

RE sites; not present (what type of cloning requires RE sites not present in the gene of interest)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

In pET28 cloning, for an N-term his-tag, how would you design a forward primer?

A

Forward Primer: 5’ (Junk) (Nde1) (Gene) 3 (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

In pET28 cloning, for an N-term his-tag, how would you design a reverse primer?

A

Reverse: (gene) (stop codon) (restriction site) (Junk) –> Reverse complement: (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

In pET28 cloning, for no his-tag, how would you design a reverse primer?

A

Reverse: (gene) (stop codon) (restriction site) (Junk) –> Reverse complement: (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

In pET28 cloning, for no his-tag, how would you design a forward primer?

A

Forward Primer: 5’ (Junk) (Nco1) (extra bps to make the sequence in-frame) (Gene) 3’ (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

In pET28 cloning, for a C-term his-tag, how would you design a forward primer?

A

Forward Primer: 5’ (Junk) (Nco1) (extra bps to make the sequence in-frame) (Gene) 3’ (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

In pET28 cloning, for a C-term his-tag, how would you design a reverse primer?

A

Reverse: (gene) (restriction site) (Junk) –> Reverse complement: (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

What are these advantages of RE Cloning Sticky End

A
  • High ligation efficiency
  • Directional insertion
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

What are these disadvantages of RE Cloning Sticky End

A
  • time-consuming
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

What are these disadvantages of RE Cloning Blunt Ended

A
  • Not efficient ligation
  • Non-direction
  • Time consuming
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
47
Q

__________ cleaves peptidoglycan bonds and degrades cell wall

A

Lysozyme (function)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
48
Q

__________ cause disintegration of membranes and can be very useful for G- bacteria

A

Detergents (function)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
49
Q

Combination of __________and __________ enhance cell lysis

A

detergents and lysozyme (these in tandem enhance what)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
50
Q

__________ inhibits proteases

A

PMSF (function)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
51
Q

What is used for protein protection (2)

A
  • PMSF
  • Lower temps (~4C)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
52
Q

As salt concentration increases, protein solubility __________. The net effect is dehydration of proteins which promotes __________.

A

decreases; self-association and aggregation (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
53
Q

AdhP is expressed in the ___________ vector

A

pET101D (what notable enzyme is expressed here)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
54
Q

For the pET101D vector the ___________ is what allows for purification using an Ni-NTA column

A

C-term his-tag (function in pET101D)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
55
Q

For the pET101D vector the __________ is what allows for detection on the Western Blot

A

V5 epitope (function in pET101D)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
56
Q

The Bradford Assay utilizes ____________

A

Coomassie Blue (used in what)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
57
Q

__________ is used to check the purity and MW of our product

A

SDS-PAGE (uses)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
58
Q

SDS-PAGE separates by __________

A

size (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
59
Q

For SDS-PAGE the negatively charged molecules move toward the __________ and the positively charged molecules move toward the __________.

A

anode; cathode (s)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
60
Q

__________ can be used to separate aa’s, DNA, RNA, and can be used for DNA sequencing.

A

SDS-PAGE (things this can separate)

61
Q

Twice as many bubbles arise from the __________, because this is where __________ takes place

A

cathodes; reduction (s)

62
Q

pH increases in the ___________ chamber

A

cathode (s)

63
Q

TEMED is the catalyst for __________ to create __________

A

cleaving APS; sulfate radicals (what cleaves APS to create the radicals)

64
Q

The ratio used for the gel indicates the amount of ____________

A

acrylamide to BIS (what does this denote in a gel)

65
Q

Lower the concentrations of acrylamide, larger are the pores in the gel, allowing analysis of __________ biomolecules.

A

higher-molecular weight (s)

66
Q

SDS is used for __________

A

denaturing (s)

67
Q

The sample buffer/loading dye includes (4)

A

SDS, pH 6.8, reducing agent, and color (these are components of what)

68
Q

Denatured proteins bind to ~1.4 g SDS/g protein, or ~one SDS molecule for every two amino acids imparting __________ for all the proteins

A

uniform mass/charge ratio (how is this created in SDS-PAGE)

69
Q

Mercaptoethanol is a(n) __________

A

reducing agent (s)

70
Q

Cysteine-cysteine disulfide bonds (covalent bond) can be broken only by using reducing agents such as __________ or __________.

A

DTT or 2-mercaptoethanol (what are these)

71
Q

The sample buffer used for SDS-PAGE contains a tracking dye, __________, which will migrate with the __________ of the proteins being separated on the gel.

A

bromophenol blue (BPB); leading edge (what is this used in and what does it do)

72
Q

Protein can be visualized using __________ staining. The dye is positively charged and binds to the negatively charged protein.

A

Coomassie Brilliant Blue R-250 (what is this used for)

73
Q

Discontinuous Electrophoresis is used for SDS-PAGE due to running buffer having pH of __________ –> stacking having pH of __________ –> resolving having pH of __________

A

8.3; 6.8; 8.8 (s)

74
Q

Glycine is negative in the __________

A

resolving gel (charge of glycine here)

75
Q

In the __________ glycine molecules are neutral chloride and protein are negative. Protein is in between Cl- and glycine

A

stacking gel (charge of glycine and location of protein, glycine, and chlorine)

76
Q

In the stacking gel, negatively charged protein molecules move forward in the field rapidly through the large pores of 4% gel, and __________

A

stack on each other (s)

77
Q

In the stacking gel different charge status of different molecules creates a potential difference, leading to a __________ in the electric field

A

localized increase (what causes the localized increase in the stacking gel)

78
Q

In the __________ glycine, chloride and protein, all are negatively charged - and hence uniform charge through-out the gel

A

resolving gel (charges of glycine, chlorine, and protein

79
Q

In the __________ molecules are separated based on their sizes

A

resolving gel (how are molecules separated here)

80
Q

The __________ of SDS-PAGE- is because of discontinuity between the stacking and running/resolving gels

A

resolving power (what produces this in SDS-PAGE)

81
Q

A standard curve of the __________ versus __________ of protein standards to determine MW of unknown protein by SDS-PAGE

A

log (MW); Rf (What can these be used to create in SDS-PAGE and how)

82
Q

The western blot (also called as immunoblot), takes advantage of the highly specific interaction of __________ to an __________ to detect specific proteins in a samples including those in tissue/cell extracts. It is a widely used analytical technique in molecular biology and immunology.

A

antigen to an antibody (what is western blot used for)

83
Q

A(n) __________ is a molecule that can induce an immune response

A

antigen (define)

84
Q

A(n) __________ is a protein (immunoglobulin) produced in response to antigen

A

antibody

85
Q

Each antigen has distinct surface features, or epitopes, which is recognized by the antibody, this is called the __________

A

epitope

86
Q

The __________ is a region on an antibody that binds to antigens.

A

fragment antigen-binding (Fab) (define)

87
Q

__________ composition will be identical for all the antibodies produced in an organism

A

Fragment crystallizable (Fc) (relation to antibodies)

88
Q

What are these advantages of Wet Transfer

A
  • Useful for gaining quantitative information
  • Highly customizable in terms of: temperature, voltage, and buffer to achieve complete transfer
89
Q

What are these disadvantages of Wet Transfer

A
  • Time consuming
  • Large amounts of buffer and excess heat generation (requires running in cold-rooms or ice-packs)
90
Q

What are these advantages of Dry, Semi-dry transfer

A

Conserves time and reagents

91
Q

What are the disadvantages of Dry, Semi dry transfer

A
  • May not allow quantitative detection for all proteins
  • Especially those that are very small or very large
  • The stack can dry out resulting in improper transfers
92
Q

What are these advantages of Nitrocellulose

A
  • Has a good affinity for protein and therefore has good retention.
  • Cheaper compared to other membranes
93
Q

What are these disadvantages of Nitrocellulose

A
  • is fragile and is not recommended for stripping and re-probing.
94
Q

What are these advantages of PVFD

A
  • Has higher binding capacity than other option
  • Offer higher mechanical strength and allow for reprobing and storage
  • Allows for the analysis of hydrophobic proteins.
95
Q

What are these disadvantages of PVFD

A
  • Background staining can be higher and hence careful washing is required
96
Q

Blocking is a very important step in western blotting to prevent:

A

antibodies from binding to the membrane nonspecifically (what prevents this)

97
Q

In Western Blotting the membrane has more exposed surface than there are proteins in the gel. The exposed regions of the membrane can bind to antibody __________

A

non-specifically (s)

98
Q

Blocking is often made with __________ or __________ in TBST/PBST buffer [a mixture of tris buffered saline (TBS) or phosphate buffered saline (PBS) and Tween 20].

A

BSA or nonfat dried milk (what are these used for)

99
Q

What are these advantages of Non-fat milk powder

A
  • Cheap compared to alt.
  • Readily available and easy to prepare from powder.
100
Q

What are these disadvantages of Non-fat milk powder

A
  • Shouldn’t be used to detect phosphorylated proteins.
  • Should not be used if avidinbiotin detection systems are being used as it contains biotin.
101
Q

What are these advantages of BSA

A
  • Good general blocking agent.
  • Clearer results as it is just one protein, and less cross reactions with the antibody.
  • Works for phosphorylated
    proteins
102
Q

What are these disadvantages of BSA

A
  • More expensive than alt.
  • Not compatible with lectin probes as it contains carbohydrates that can increase background.
103
Q

In __________ the primary antibody is directly conjugated to an enzyme that will catalyze a calorimetric/fluorescent reaction

A

direct detection (define)

104
Q

For __________, two different antibodies are used in sequence for the detection step. First, the Western blot is incubated with an unlabeled primary antibody directed against the target protein. After washing, a labeled secondary antibody is used to detect the presence of the primary antibody, and thus the target protein.

A

indirect detection (define)

105
Q

In the Western Blot of AdhP the primary antibody, __________ is directly conjugated to the _____________.

A

Anti-V5; HRP enzyme (how are these related in Western Blot)

106
Q

__________ allows detection of recombinant proteins containing the V5 epitope.

A

Anti-V5-HRP antibody (what does this allow for)

107
Q

HRP uses __________ to catalyze the oxidation of a variety of organic compounds, many of which have been developed for detection as colored compounds

A

H2O2 (s)

108
Q

HRP (horse radish peroxidase) is a heme containing oxidoreductase enzyme catalyzes oxidation of 4-chloro-1-naphthol (4CN) in the presence of hydrogen peroxide. This leads to the formation of an insoluble __________- that gets deposited on the membrane where the antibody is present

A

purple colored quinone product (what is this a product of)

109
Q

__________ utilizes an enzyme system to show specific interaction of an antigen with its antibody. It is a method of quantifying an antigen/antibody immobilized on a solid surface.

A

Enzyme-linked immunosorbent assay (ELISA) (main function compared to Western Blot)

110
Q

__________ is used as diagnostic tools in medicine such as HIV test, Lyme’s disease test, COVD-19 rapid serology antibody test, Strep-test, and pregnancy test (hCG) etc

A

ELISA (can be used as a diagnostic for what)

111
Q

__________ is a pH gradient agarose or polyacrylamide gel copolymerized with a mixture of ampholytes (molecule that can act both as acid and base).

A

IEF gel (what is this made up of)

112
Q

At __________, in IEF, the proteins are positively charged, so they will migrate towards the cathode (negatively charged electrode).

A

pH < pI (what does this result in in IEF)

113
Q

In __________, as it migrates through a gradient of increasing pH, the protein’s overall charge will decrease until the protein reaches the pH region that corresponds to its pI (where its’ net charge = 0) and so there is no electrical attraction towards the electrode.

A

IEF

114
Q

IEF separates gel based on their __________

A

pI (this is the separating principle in what)

115
Q

In IEF SDS is NOT used- if there is need for denaturation, __________ is used to unfold the protein.

A

urea (what does this do in IEF)

116
Q

What are these uses of IEF

A

-Compare protein expression profile under different growth conditions
-Changes in post-translational modifications of a protein

117
Q

Enzymes accelerate rate of a specific reaction by lowering its __________

A

activation energy (relation to enzymes)

118
Q

Enzymes do not alter the __________, __________, and are not __________ during the rxn

A

thermodynamics, reaction, consumed (relation to enzymes)

119
Q

_____________ is an intermediate state during a chemical reaction that has a higher energy than the reactants or the products.

A

Transition state (define)

120
Q

__________ is the energy required for the reactants to reach the transition state

A

Activation energy (EA) (define)

121
Q

Enzymes are affected by (4)

A

Affected by Temp, pH, cofactors and coenzymes, and enzymatic inhibitors (what do these affect)

122
Q

For enzymes higher temps are generally better because they allow for more __________

A

collisions (what causes an increase in these)

123
Q

Optimal pH for enzymes is __________

A

between 6-9 (what is this range for)

124
Q

Cofactors can be (4)

A

Can be loosely bound metal ions, loosely bound organic molecules, and tightly bounds organic and inorganic molecules

125
Q

Types of cofactors (2)

A
  • Coenzymes
  • Prosthetic groups
126
Q

__________ are the non-protein organic molecules needed for enzyme activity, bound loosely to the enzyme are coenzymes

A

Coenzymes (define)

127
Q

____________ are the non-protein organic or inorganic molecules needed for enzyme activity, bound tightly (even via covalent bonds) to an enzyme are prosthetic group

A

Prosthetic groups (define)

128
Q

Active site: A small region of an enzyme, where the __________ binds and the reaction/catalysis takes place

A

substrate (relation to active site)

129
Q

The __________ is the molecule and enzyme works on

A

substrate (define)

130
Q

__________ is the amount of enzyme needed to convert a unit mole of a substrate to product per unit time under standard reaction conditions.

A

Enzyme activity (define)

131
Q

1 Unit : The amount of enzyme needed to convert one __________ of substrate to product in __________ under standard reaction conditions (a given pH and temperature).

A

micromole; one minute (s)

132
Q

__________: activity/mL multiplied by total volume of the enzyme

A

Total activity (define)

133
Q

__________: activity of an enzyme per mg, obtained by dividing activity by total protein (unit/mg)

A

Specific activity (define)

134
Q

__________: Divide specific activity of a particular step of purification by the specific activity of the lysate

A

Fold purification (define)

135
Q

__________: Divide the total activity of a particular step of purification by the total activity of the lysate and take the %

A

Percent yield (define)

136
Q

The __________ of a reaction is the change in concentration of a reactant/product per unit time. –amount of product formed, or reactant consumed (in concentration units) per unit time.

A

speed/rate (define in relation to kinetics)

137
Q

Slope of ABS v. Time of a reaction indicates the __________

A

velocity (define in relation to kinetics)

138
Q

The rate of reaction increases as __________ increases

A

substrate concentration (if this increases, what also increases)

139
Q

Maximum activity occurs when:

A

the enzyme is saturated with substrates (what does this lead to)

140
Q

__________ is the number of cycles an enzyme completes in 1s; The speed at which an enzymes catalyzes a reaction

A

Kcat (define)

141
Q

__________ indicates how well the substrate can bind an enzyme, the lower the number the better binding

A

Km (define)

142
Q

The __________ is the efficiency of an enzyme for a particular substrate

A

Specificity constant (Kcat/Km) (define)

143
Q

The rate of a reaction is directly proportional to the __________ of each reactant raised to a power.

A

concentration (s)

144
Q

The __________ (n and m) on each reactant in the rate law is called the order with respect to that reactant.

A

exponent (s)

145
Q

The GTGG overhang promotes __________

A

annealing (what causes this in pET101D cloning)

146
Q

pET101D has __________ resistance

A

ampicillin (what type of cloning has resistance to this)

147
Q

T7 polymerase is required for expression of __________ in pET101D

A

recombinant protein (what is required for the expression of this in pET101D cloning)

148
Q

TA cloning is (directional/non-directional)

A

non-directional (s)

biochemical methods (2 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions