exam 2 Flashcards
(148 cards)
1
Q
__________ relaxes supercoiled DNA
A
DNA gyrase (function)
2
Q
__________ breaks hydrogen bonds to make DNA single-stranded
A
DNA helicase (function)
3
Q
DNA Polymerase removes errors by a __________ activity
A
3’-5’ exonuclease (what uses this)
4
Q
Isopropanol allows for __________; Addition of __________ can also aid in this
A
DNA precipitation; salt (s)
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)
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)
7
Q
Ideal primer length is __________
A
20-30BP (what is this range for)
8
Q
In PCR, the product which has the desired gene region appears only in the _________
A
third cycle (s)
9
Q
The reverse primer is the reverse complement of the __________
A
end of the coding strand (s)
10
Q
What direction does DNA synthesis happen?
A
5’ to 3’ (s)
11
Q
Good primer Tm is between __________
A
52-65C (what is this range for)
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)
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)
14
Q
Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to __________
A
LOG(MW) (s)
15
Q
Plasmids can replicate __________
A
independently (s)
16
Q
__________ are modified plasmids used in recombinant DNA technology for cloning.
A
Vectors (define)
17
Q
A reporter gene is a __________ gene
A
drug resistance (s)
18
Q
The __________ is where the gene can be inserted.
A
MCS (s)
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)
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)
21
Q
LacO is an Operator/DNA (regulatory) sequence that controls the __________ in TOPO cloning
A
protein expression (what controls this in TOPO cloning)
22
Q
LacI protein binds to LacO and prevents activity of __________.
A
T7 RNA polymerase (what prevents this)
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)
24
Q
TOPO cloning includes a V5 epitope and c-term his-tag, if you do not include the __________
A
STOP codon (s)
25
What are these advantages of TOPO Cloning
- Doesn't require restriction digest
- Directional insertion
- No ligation required
- Relatively time saving
26
What are these disadvantages of TOPO Cloning
- Overhangs sensitive to degradation
- Must purchase a vector each time
27
TA cloning uses a Taq polymerase that adds __________ to the __________ of the PCR product.
A; 3' end (what adds A to the 3' end of PCR product)
28
What are these steps for Plasmid Purification
- Grow cells in abx, then pellet
- Resuspend
- Lyse
- Neutralize pH
- Capture plasmid DNA, remove excess salt
- Elute
29
Number of double stranded DNA copies that span the size between the primers after n cycles of PCR:
(2^n - (n*2)) (what does this eqn represent)
30
__________ are endonucleases commonly found in bacteria or archaea and they provide defense mechanism against foreign DNA
Restriction Enzymes
31
Restriction enzymes have specific recognition sequences and cleavage site, and they cut DNA sequences in a __________ manner
predictable (s)
32
The restriction enzyme recognition sequences are __________
self-complementary (what sequences are self-complementary)
33
RNA and DNA sequences that code for protein are called __________
open reading frames (ORF) (define)
34
Each RNA/DNA strand has __________ ORF's
3 (s)
35
What are these advantages of TA Cloning
- Doesn't require restriction enzymes
36
What are these disadvantages of TA Cloning
- Vector and insert sensitive to degradation
- Non-directional cloning
- Taq-polymerase has low proofreading ability
37
For deciding the vector for pET28 cloning, choose two different __________ that are __________ within your gene of interest
RE sites; not present (what type of cloning requires RE sites not present in the gene of interest)
38
In pET28 cloning, for an N-term his-tag, how would you design a forward primer?
Forward Primer: 5' (Junk) (Nde1) (Gene) 3 (s)
39
In pET28 cloning, for an N-term his-tag, how would you design a reverse primer?
Reverse: (gene) (stop codon) (restriction site) (Junk) --> Reverse complement: (s)
40
In pET28 cloning, for no his-tag, how would you design a reverse primer?
Reverse: (gene) (stop codon) (restriction site) (Junk) --> Reverse complement: (s)
41
In pET28 cloning, for no his-tag, how would you design a forward primer?
Forward Primer: 5' (Junk) (Nco1) (extra bps to make the sequence in-frame) (Gene) 3' (s)
42
In pET28 cloning, for a C-term his-tag, how would you design a forward primer?
Forward Primer: 5' (Junk) (Nco1) (extra bps to make the sequence in-frame) (Gene) 3' (s)
43
In pET28 cloning, for a C-term his-tag, how would you design a reverse primer?
Reverse: (gene) (restriction site) (Junk) --> Reverse complement: (s)
44
What are these advantages of RE Cloning Sticky End
- High ligation efficiency
- Directional insertion
45
What are these disadvantages of RE Cloning Sticky End
- time-consuming
46
What are these disadvantages of RE Cloning Blunt Ended
- Not efficient ligation
- Non-direction
- Time consuming
47
__________ cleaves peptidoglycan bonds and degrades cell wall
Lysozyme (function)
48
__________ cause disintegration of membranes and can be very useful for G- bacteria
Detergents (function)
49
Combination of __________and __________ enhance cell lysis
detergents and lysozyme (these in tandem enhance what)
50
__________ inhibits proteases
PMSF (function)
51
What is used for protein protection (2)
- PMSF
- Lower temps (~4C)
52
As salt concentration increases, protein solubility __________. The net effect is dehydration of proteins which promotes __________.
decreases; self-association and aggregation (s)
53
AdhP is expressed in the ___________ vector
pET101D (what notable enzyme is expressed here)
54
For the pET101D vector the ___________ is what allows for purification using an Ni-NTA column
C-term his-tag (function in pET101D)
55
For the pET101D vector the __________ is what allows for detection on the Western Blot
V5 epitope (function in pET101D)
56
The Bradford Assay utilizes ____________
Coomassie Blue (used in what)
57
__________ is used to check the purity and MW of our product
SDS-PAGE (uses)
58
SDS-PAGE separates by __________
size (s)
59
For SDS-PAGE the negatively charged molecules move toward the __________ and the positively charged molecules move toward the __________.
anode; cathode (s)
60
__________ can be used to separate aa's, DNA, RNA, and can be used for DNA sequencing.
SDS-PAGE (things this can separate)
61
Twice as many bubbles arise from the __________, because this is where __________ takes place
cathodes; reduction (s)
62
pH increases in the ___________ chamber
cathode (s)
63
TEMED is the catalyst for __________ to create __________
cleaving APS; sulfate radicals (what cleaves APS to create the radicals)
64
The ratio used for the gel indicates the amount of ____________
acrylamide to BIS (what does this denote in a gel)
65
Lower the concentrations of acrylamide, larger are the pores in the gel, allowing analysis of __________ biomolecules.
higher-molecular weight (s)
66
SDS is used for __________
denaturing (s)
67
The sample buffer/loading dye includes (4)
SDS, pH 6.8, reducing agent, and color (these are components of what)
68
Denatured proteins bind to ~1.4 g SDS/g protein, or ~one SDS molecule for every two amino acids imparting __________ for all the proteins
uniform mass/charge ratio (how is this created in SDS-PAGE)
69
Mercaptoethanol is a(n) __________
reducing agent (s)
70
Cysteine-cysteine disulfide bonds (covalent bond) can be broken only by using reducing agents such as __________ or __________.
DTT or 2-mercaptoethanol (what are these)
71
The sample buffer used for SDS-PAGE contains a tracking dye, __________, which will migrate with the __________ of the proteins being separated on the gel.
bromophenol blue (BPB); leading edge (what is this used in and what does it do)
72
Protein can be visualized using __________ staining. The dye is positively charged and binds to the negatively charged protein.
Coomassie Brilliant Blue R-250 (what is this used for)
73
Discontinuous Electrophoresis is used for SDS-PAGE due to running buffer having pH of __________ --> stacking having pH of __________ --> resolving having pH of __________
8.3; 6.8; 8.8 (s)
74
Glycine is negative in the __________
resolving gel (charge of glycine here)
75
In the __________ glycine molecules are neutral chloride and protein are negative. Protein is in between Cl- and glycine
stacking gel (charge of glycine and location of protein, glycine, and chlorine)
76
In the stacking gel, negatively charged protein molecules move forward in the field rapidly through the large pores of 4% gel, and __________
stack on each other (s)
77
In the stacking gel different charge status of different molecules creates a potential difference, leading to a __________ in the electric field
localized increase (what causes the localized increase in the stacking gel)
78
In the __________ glycine, chloride and protein, all are negatively charged - and hence uniform charge through-out the gel
resolving gel (charges of glycine, chlorine, and protein
79
In the __________ molecules are separated based on their sizes
resolving gel (how are molecules separated here)
80
The __________ of SDS-PAGE- is because of discontinuity between the stacking and running/resolving gels
resolving power (what produces this in SDS-PAGE)
81
A standard curve of the __________ versus __________ of protein standards to determine MW of unknown protein by SDS-PAGE
log (MW); Rf (What can these be used to create in SDS-PAGE and how)
82
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.
antigen to an antibody (what is western blot used for)
83
A(n) __________ is a molecule that can induce an immune response
antigen (define)
84
A(n) __________ is a protein (immunoglobulin) produced in response to antigen
antibody
85
Each antigen has distinct surface features, or epitopes, which is recognized by the antibody, this is called the __________
epitope
86
The __________ is a region on an antibody that binds to antigens.
fragment antigen-binding (Fab) (define)
87
__________ composition will be identical for all the antibodies produced in an organism
Fragment crystallizable (Fc) (relation to antibodies)
88
What are these advantages of Wet Transfer
- Useful for gaining quantitative information
- Highly customizable in terms of: temperature, voltage, and buffer to achieve complete transfer
89
What are these disadvantages of Wet Transfer
- Time consuming
- Large amounts of buffer and excess heat generation (requires running in cold-rooms or ice-packs)
90
What are these advantages of Dry, Semi-dry transfer
Conserves time and reagents
91
What are the disadvantages of Dry, Semi dry transfer
- 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
What are these advantages of Nitrocellulose
- Has a good affinity for protein and therefore has good retention.
- Cheaper compared to other membranes
93
What are these disadvantages of Nitrocellulose
- is fragile and is not recommended for stripping and re-probing.
94
What are these advantages of PVFD
- 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
What are these disadvantages of PVFD
- Background staining can be higher and hence careful washing is required
96
Blocking is a very important step in western blotting to prevent:
antibodies from binding to the membrane nonspecifically (what prevents this)
97
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 __________
non-specifically (s)
98
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].
BSA or nonfat dried milk (what are these used for)
99
What are these advantages of Non-fat milk powder
* Cheap compared to alt.
* Readily available and easy to prepare from powder.
100
What are these disadvantages of Non-fat milk powder
* Shouldn't be used to detect phosphorylated proteins.
* Should not be used if avidinbiotin detection systems are being used as it contains biotin.
101
What are these advantages of BSA
* Good general blocking agent.
* Clearer results as it is just one protein, and less cross reactions with the antibody.
* Works for phosphorylated
proteins
102
What are these disadvantages of BSA
* More expensive than alt.
* Not compatible with lectin probes as it contains carbohydrates that can increase background.
103
In __________ the primary antibody is directly conjugated to an enzyme that will catalyze a calorimetric/fluorescent reaction
direct detection (define)
104
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.
indirect detection (define)
105
In the Western Blot of AdhP the primary antibody, __________ is directly conjugated to the _____________.
Anti-V5; HRP enzyme (how are these related in Western Blot)
106
__________ allows detection of recombinant proteins containing the V5 epitope.
Anti-V5-HRP antibody (what does this allow for)
107
HRP uses __________ to catalyze the oxidation of a variety of organic compounds, many of which have been developed for detection as colored compounds
H2O2 (s)
108
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
purple colored quinone product (what is this a product of)
109
__________ 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.
Enzyme-linked immunosorbent assay (ELISA) (main function compared to Western Blot)
110
__________ 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
ELISA (can be used as a diagnostic for what)
111
__________ is a pH gradient agarose or polyacrylamide gel copolymerized with a mixture of ampholytes (molecule that can act both as acid and base).
IEF gel (what is this made up of)
112
At __________, in IEF, the proteins are positively charged, so they will migrate towards the cathode (negatively charged electrode).
pH < pI (what does this result in in IEF)
113
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.
IEF
114
IEF separates gel based on their __________
pI (this is the separating principle in what)
115
In IEF SDS is NOT used- if there is need for denaturation, __________ is used to unfold the protein.
urea (what does this do in IEF)
116
What are these uses of IEF
-Compare protein expression profile under different growth conditions
-Changes in post-translational modifications of a protein
117
Enzymes accelerate rate of a specific reaction by lowering its __________
activation energy (relation to enzymes)
118
Enzymes do not alter the __________, __________, and are not __________ during the rxn
thermodynamics, reaction, consumed (relation to enzymes)
119
_____________ is an intermediate state during a chemical reaction that has a higher energy than the reactants or the products.
Transition state (define)
120
__________ is the energy required for the reactants to reach the transition state
Activation energy (EA) (define)
121
Enzymes are affected by (4)
Affected by Temp, pH, cofactors and coenzymes, and enzymatic inhibitors (what do these affect)
122
For enzymes higher temps are generally better because they allow for more __________
collisions (what causes an increase in these)
123
Optimal pH for enzymes is __________
between 6-9 (what is this range for)
124
Cofactors can be (4)
Can be loosely bound metal ions, loosely bound organic molecules, and tightly bounds organic and inorganic molecules
125
Types of cofactors (2)
- Coenzymes
- Prosthetic groups
126
__________ are the non-protein organic molecules needed for enzyme activity, bound loosely to the enzyme are coenzymes
Coenzymes (define)
127
____________ are the non-protein organic or inorganic molecules needed for enzyme activity, bound tightly (even via covalent bonds) to an enzyme are prosthetic group
Prosthetic groups (define)
128
Active site: A small region of an enzyme, where the __________ binds and the reaction/catalysis takes place
substrate (relation to active site)
129
The __________ is the molecule and enzyme works on
substrate (define)
130
__________ is the amount of enzyme needed to convert a unit mole of a substrate to product per unit time under standard reaction conditions.
Enzyme activity (define)
131
1 Unit : The amount of enzyme needed to convert one __________ of substrate to product in __________ under standard reaction conditions (a given pH and temperature).
micromole; one minute (s)
132
__________: activity/mL multiplied by total volume of the enzyme
Total activity (define)
133
__________: activity of an enzyme per mg, obtained by dividing activity by total protein (unit/mg)
Specific activity (define)
134
__________: Divide specific activity of a particular step of purification by the specific activity of the lysate
Fold purification (define)
135
__________: Divide the total activity of a particular step of purification by the total activity of the lysate and take the %
Percent yield (define)
136
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.
speed/rate (define in relation to kinetics)
137
Slope of ABS v. Time of a reaction indicates the __________
velocity (define in relation to kinetics)
138
The rate of reaction increases as __________ increases
substrate concentration (if this increases, what also increases)
139
Maximum activity occurs when:
the enzyme is saturated with substrates (what does this lead to)
140
__________ is the number of cycles an enzyme completes in 1s; The speed at which an enzymes catalyzes a reaction
Kcat (define)
141
__________ indicates how well the substrate can bind an enzyme, the lower the number the better binding
Km (define)
142
The __________ is the efficiency of an enzyme for a particular substrate
Specificity constant (Kcat/Km) (define)
143
The rate of a reaction is directly proportional to the __________ of each reactant raised to a power.
concentration (s)
144
The __________ (n and m) on each reactant in the rate law is called the order with respect to that reactant.
exponent (s)
145
The GTGG overhang promotes __________
annealing (what causes this in pET101D cloning)
146
pET101D has __________ resistance
ampicillin (what type of cloning has resistance to this)
147
T7 polymerase is required for expression of __________ in pET101D
recombinant protein (what is required for the expression of this in pET101D cloning)
148
TA cloning is (directional/non-directional)
non-directional (s)
