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Biochemical Methods > Exam II > Flashcards

Exam II Flashcards

1
Q

What are the steps of gene cloning?

A
  1. Isolation of plasmid DNA and DNA containing gene of interest
  2. Gene inserted into plasmid
  3. Plasmid put into bacterial cell
  4. Cells cloned with gene of interest
  5. Identification of desired clone
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2
Q

What was our gene of interest that we cloned using TOPO?

A

adhP

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3
Q

What does the gene adhP encode for?

A

Alcohol dehydrogenase

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4
Q

What method is used to get the plasmid back into a host cell?

A

Transformation

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5
Q

What is the plasmid we used in lab called?

A

pET-101D/TOPO

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6
Q

How will we purifiy our cloned proteins in lab?

A

Affinity Chromatography

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7
Q

What does PCR stand for?

A

polymerase chain reaction

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8
Q

What are two ways cloning can be achieved?

A
  1. take a small portion of organisms and allowing it to grow into a full organism (carrot)
  2. Embryo Splitting
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9
Q

What is a fundamental process of copying the DNA which occurs in all living organisms and is the basis for biological inheritance?

A

DNA replication

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10
Q

What is the difference between DNA and RNA?

A
  1. DNA is double stranded, with two anti-parallel strands

2. RNA is a single strand and can form secondary and tertiary structures (tRNA)

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11
Q

What are the components of a nucleotide?

A

Base
Sugar
Phosphate

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12
Q

The base can either be a purine or a what?

A

pyrimidine

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13
Q

Adenine and guanine are what type of base?

A

purine

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14
Q

Thymine/Uracil and cytosine are what type of base?

A

pyrimidine

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15
Q

Guanine and cytosine form how many hydrogen bonds?

A

3

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16
Q

Thymine/Uracil and adenine form how many hydrogen bonds?

A

2

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17
Q

The side chain group on the 2’ Carbon on DNA is what?

A

H

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18
Q

The side chain group on the 2’ Carbon on RNA is what?

A

OH

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19
Q

What group is on the 3’ Carbon for both RNA and DNA?

A

OH

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20
Q

The phosphate backbone attaches to which carbons of both DNA and RNA?

A

5’

3’

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21
Q

What is ligation?

A

When a phosphate group attaches to the 3’ or 5’ carbon of a sugar

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22
Q

What serves as the template for the reproduction of the complementary strand?

A

Each strand of the original double stranded DNA molecule

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23
Q

What are produced from a single double stranded DNA molecule?

A

Two identical DNA molecules

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24
Q

Which strand is read 3’ to 5’ by the DNA polymerase and produces a continuously growing strand?

A

Leading strand

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25
Q

Which strand is read from 3’ to 5’ but produces short strands that must be linked together?

A

Lagging Strand

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26
Q

What is located at the replication fork and separates the lagging and leading strands?

A

Helicase

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27
Q

Who discovered DNA polymerase in 1956?

A

Arthur Kornberg (Nobel Prize in 1959)

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28
Q

What can only add nucleotides to the 3’ end of the primer- extending the chain in the 5’ to 3’ direction?

A

DNA polymerase

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29
Q

What is the error rate of DNA polymerase?

A

1 in every billion base pair added

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30
Q

What are they short strands that are formed from the lagging strand called?

A

Okazaki fragments

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31
Q

PCR is a process that imitates what?

A

the natural DNA replication

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32
Q

The membranes of bacterial cells are made up of what?

A

lipids

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33
Q

The cell walls of bacterial cells are made up of what?

A

sugars

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34
Q

What kind of bacteria has both an inner and outer cell wall?

A

Gram (-) Bacteria

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35
Q

Gram positive bacteria have a thick layer of what?

A

peptidoglycan

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36
Q

Gram negative bacteria have what kind of peptidoglycan layer?

A

thin

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37
Q

What are plasmids?

A

Non-chromosomal DNA

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38
Q

Does the replication of plasmids depend on the replication of the chromosome?

A

No

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39
Q

Are there many copies of plasmids within a cell?

A

Yes

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40
Q

What are the steps of genomic DNA purification?

A
  1. Lyse all cells
  2. Denature proteins
  3. Inactivate endogenous nucleases
  4. Isolate/ Purify genomic DNA
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41
Q

What does Tide Free 2X laundry detergent contain?

A

proteases
lipases
glycolytic enzymes

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42
Q

What is the function of the proteases in Tide?

A

degrades proteins

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43
Q

What is the function of the lipases in Tide?

A

cleaves fat/lipids

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44
Q

What is the function of the glycolytic enzymes in Tide?

A

degrade sugar

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45
Q

What has limited solubility in water; thus forms a separate organic phase from the aqueous phase?

A

n-butonal

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46
Q

What do you do to separate the cell debris, organic phase, and inorganic phase?

A

Spin the sample

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47
Q

Which phase is removed and transferred to a new tube after the n-butanol is added and the tube is spun?

A

The aqueous phase (inorganic)

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48
Q

Which phase will the DNA and RNA go to and why?

A

aqueous (inorganic). They are starches

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49
Q

Why do you add 2-propanol to the aqueous layer?

A

To precipitate the genomic DNA and RNA

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50
Q

Why do you wash with 70% ethanol?

A

To remove salt contamination

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51
Q

why is it crucial that you dry out the ethanol before re-dissolving the DNA pellet back in water?

A

maintains the activity of enzymes used further down in the protocol.

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52
Q

What is added to remove RNA contamination?

A

RNase A (incubate at 60 C)

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53
Q

What are the components of PCR?

A 
two primer strands (forward and reverse)
dNTPs
template DNA
appropriate buffer
DNA polymerase
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54
Q

Who invented PCR?

A

Kary Mullis (nobel Prize 1993)

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55
Q

What are other names for the coding strand?

A

sense strand

positive strand

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56
Q

What are other names for the complementary strand?

A

anti-sense strand

negative strand

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57
Q

What should be the length of the primers?

A

20 to 30 nucleotides long

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58
Q

What is the denaturation step of PCR?

A

DNA is heated to separate strands 95 C

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59
Q

What is the annealing step of PCR?

A

DNA strands are cooled to allow them to anneal with the primers (52-65 C)

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60
Q

What is the extension step of PCR?

A

Temperature is increased to ~72 C and DNA Polymerase completes the DNA strand

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61
Q

What direction does synthesis/replication occur?

A

5’ to 3’

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62
Q

How many cycles can a PCR reaction run for?

A

25-35 cycles

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63
Q

A PCR reaction with N cycles will give how many DNA molecules?

A

2^N

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64
Q

What has the same sequence as the “coding” strand and anneals with the complement strand?

A

FWD primer

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65
Q

What has the same sequence as the complement strand and anneals with the coding strand?

A

REV primer

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66
Q

What determines the specificity and significantly affects the ability for the primer to anneal to the template?

A

primer length

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67
Q

If the primer is too short what occurs?

A

low specificity, resulting in non-specific amplification

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68
Q

If the primer is too long what occurs?

A

decreases the template-binding efficiency at normal annealing temperatures due to the higher probability of forming secondary structures such as hairpins

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69
Q

What are some examples of secondary structures?

A

hairpins
self-Dimer
Dimer

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70
Q

How do you determine the annealing temperature?

A

Should be a few degrees lower than the melting temperatures of the primers which is based on the number of GC and AT pairs

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71
Q

What is the formula to estimate the Tm?

A

Tm = 4 (G+C) + 2 (A+T) units are in degrees Celsius

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72
Q

What is a device that controls incubation temperatures and times?

A

Thermal Cycler

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73
Q

For how long is the denaturing step?

A

15 to 30 seconds

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74
Q

How long is the elongation step?

A

1 to 2 min for every 1000 base pairs

1 min/kb at 68 to 74 C is optimal

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75
Q

How long is the elongation step?

A

5 to 10 min at 68 to 72 C

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76
Q

At what temperature should you store the genes?

A

4 C

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77
Q

When does the product that consists only of the desired regions of DNA appear?

A

3rd cycle

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78
Q

Each step is what, since two strands coming from the newly synthesized DNA can act as a template for the next reaction?

A

exponential

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79
Q

Does each strand need one primer, with opposite polarity?

A

Yes

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80
Q

What should be the ratio of absorption at 260 and absorption at 280 for DNA and proteins?

A

DNA 2

protein 0.57

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81
Q

What is the extension coefficient for ds DNA at 260nm?

A

50 micrograms/mL

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82
Q

What is the extension coefficient for ssDNA at 260 nm?

A

33 micrograms/mL

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83
Q

How long is the annealing step?

A

15 to 30 seconds

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84
Q

How do you calculate the number of DNA copies that span the size between the primers after n cycles of PCR?

A

2^n - (n*2)

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85
Q

What matrix did we use in the gel electrophoresis?

A

0.8% agarose

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86
Q

Agarose gel electrophoresis separates molecules based on what?

A

size, shape, and charge

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87
Q

What charge does DNA have?

A

negative

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88
Q

Do smaller or larger molecules move faster in gel electrophoresis?

A

smaller

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89
Q

negatively charge DNA moves toward what?

A

the anode

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90
Q

molecular size markers contain what?

A

different DNA fragments of known size

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91
Q

What buffer is usually used in electrophoresis?

A

Tris-acetate-EDTA (TAE)

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92
Q

The loading buffer contains what which allows the sample to fall into the sample wells?

A

something dense like glycerol

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93
Q

The loading buffer also contains this allowing for the visual monitoring or how far the electrophoresis has proceeded?

A

one or two tracking dyes

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94
Q

How do you pour a gel?

A 
weigh 0.8% agarose
add electrophresis buffer
microwave until completely melted
cool to 60 C
add fluorescent stain
pour into casting tray containing comb
allow to solidify
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95
Q

DNA will migrate toward the positive what?

A

electrode (anode)

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96
Q

What is intercalating mean?

A

inserts between the bases of DNA

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97
Q

what kind of dye is used?

A

fluorescent intercalating

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98
Q

What are some examples of the dye used?

A

SYBR safe or Ethidium Bromide

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99
Q

SYBR safe is how many times less toxic than Ethidium Bromide?

A

4 - 5 times

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100
Q

Do supercoiled (uncut), relaxed circular form, or linearized forms of plasmids travel the fastest?

A

supercoiled

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101
Q

Which form of plasmid travels the slowest?

A

relaxed circular form

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102
Q

What migrate through agarose gels with mobility that is inversely proportional to the log base 10 of their molecular weight?

A

linear DNA

relative mobility = 1/(log MW)

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103
Q

Typically the size of DNA bands are reported as the number of what?

A

base pairs or kilo base pairs

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104
Q

Which resolves better at low % gel, larger or smaller DNA?

A

larger 0.7%

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105
Q

Which resolves better at in 1.5% agarose, larger or smaller DNA?

A

smaller

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106
Q

What migrate proportionally faster as the voltage applied to a gel is increased?

A

larger DNA fragments

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107
Q

What is the best resolution of fragments that are greater than 2 kb?

A

5 V/cm

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108
Q

What functions do the electrophoresis buffer have?

A

establish pH
provide ions to support conductivity
maintain DNA

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109
Q

What are the potential reasons that a PCR does not work?

A

1 old/dead polymerase
2 wrong/degraded primers
3 too high concentration of primers or templates
4 wrong annealing temperature
5 ethanol (kills DNA polymerase) contamination of template DNA

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110
Q

annealing temperatures that are too low can result in what?

A

non specific priming which can lead to smears in electrophoresis

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111
Q

What is the start codon for E.coli?

A

ATG

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112
Q

What are the stop codons?

A

TAA, TAG, and TGA

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113
Q

What two amino acids have only one codon?

A

Met (ATG) and Trp (TGG)

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114
Q

Where can you retrieve the gene sequence?

A

NCBI (National Center for Biotechnology Information)

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115
Q

What is an accession number?

A

A unique identification number of a sequence. It is a string of letters and/or numbers that corresponds to a molecular sequence (protein or gen sequence)

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116
Q

What button to you select to retrieve the gene sequence?

A

FASTA

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117
Q

What is a short synthetic oligonucleotide designed to have a sequence which is the reverse complement of a region of template or target DNA?

A

a primer

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118
Q

The primer must have what to prevent fraying

A

A GC clamp

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119
Q

What is the temperature at which one half of the DNA duplex is a stable double helix, while the other half has been separated to single strand molecules?

A

Melting temperature

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120
Q

What is the optimum difference in the melting temperature of primers?

A

< 5 C (actually 3-5)

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121
Q

IDT has what tool to analyze primer?

A

Oligo analyzer

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122
Q

In addition to the melting temperature what else should be analzyed?

A

whether secondary structures are made.

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123
Q

What are modified plasmids used in recombinant DNA for cloning?

A

Vectors

124
Q

What must vectors have?

A

ORI (origin of replication)
a drug resistance gene (reporter gene)
a Multiple Cloning Site (MCS)

125
Q

What is a MCS?

A

a region in which exogenous DNA fragments can be inserted

126
Q

TOPO cloning uses DNA topoisomerase for what?

A

directed cloning

127
Q

TA cloning uses Taq polymerase that does what?

A

Adds “A” at the 3’ end of the PCR product and is non-directional

128
Q

Restriction Enzyme mediated cloning produces what two ends?

A

blunt (non-directional)

sticky (directional)

129
Q

What is an advanced method where there is no need to use restriction enzymes to produce directional clong?

A

Ligation Independent Cloning

130
Q

What antibiotic resistance gene does pET101D have?

A

ampicillian

131
Q

What two sequences does pET101D have that are essential for T7 RNA polymerase to transcribe the gene?

A

T7 promoter and T7 terminator

132
Q

What is LacO?

A

an operator sequence (regulatory) that controls the protein expression

133
Q

What is LacI

A

binds to LacO and prevent activity of T7 RNA polymerase (suppressing) expression of recombinant protein.

134
Q

What is IPTG?

A

a lactose analog that binds to LacI allowing LacI to leave the operator

135
Q

What leads to a better yield and solubility of the recombinant protein?

A

controlled expression

136
Q

What is the function of V5 eptitope?

A

codes for an I4 aa peptide

137
Q

What is used for convenient detection by recognizing the I4 aa peptide?

A

Western blot

138
Q

pET101D has a 6xHis tag at the what of the protein of interest?

A

C-terminus

139
Q

When using his tags, the primers should not contain what?

A

stop codons

140
Q

What kind of columns can bind to the his tag and purify the protein of interest?

A

Ni-NTA columns

141
Q

What is the RBS?

A

ribosome binding site

142
Q

What is the TOPO site?

A

where DNA topisomerase is covalently attached

143
Q

Where is the STOP codon on the pET101D vector?

A

after the 6xHis and V5 eptitope

144
Q

DNA topoisomerase I recognizes what, makes a cut, and then remains attached to the phosphate of the nucleotide?

A

CCTT sequence

145
Q

The 5’ end of the PCR product will have a region that matches the overhang with what sequence?

A

GTGG

146
Q

The topoisomerase promotes what?

A

the ligation of the vector and the PCR product

147
Q

Review slide 24 Lecture 13

A

Review it!

148
Q

What are some advantages of TOPO cloning?

A
  1. does not require restriction enzymes
  2. directional insertion
  3. doesnt require ligation
  4. less time required for procedure
149
Q

What are some disadvantages of TOPO cloning?

A
  1. The overhangs in the vector are sensitive to degradation

2. have to purchase vector every time from the vendor (can be costly)

150
Q

What is essential for T7 RNA polymerase to transcribe the gene?

A

T7 promoter and T7 terminator sequences

151
Q

What are endonucleases that cleave DNA strands?

A

Restriction enzymes

152
Q

What provides a defense mechanism against invading viruses?

A

Restriction enzymes

153
Q

Over 3000 what have been studied?

A

Restriction Enzymes

154
Q

In many cases the restriction site is what?

A

self complementary (palindromic)

155
Q

What happens when pET101D + adhP is cut with SacI

A

a single strand is formed

156
Q

What happens when pET101D + adhP is cut with PstI?

A

two strands are formed

157
Q

What kind of ends does SmaI create?

A

blunt ends

158
Q

What kind of ends do both EcoRI and BamHI form?

A

staggered or sticky end cuts

159
Q

What covalently attaches the vector and the insert via a phosphodiester bond between 5’ phosphate and 3’ hydroxyl?

A

ligation

160
Q

After deciding which vector you will be using, you choose two different RE sites that are not what?

A

present within your gene of interest

161
Q

Sequences must be in what?

A

frame

162
Q

When you have an N-terminal His tag where do you insert the gene?

A

after the His-tag

163
Q

Inserted junk is usually what size?

A

5 to 6 nucleotides

164
Q

What is order of information added to fwd primer in enzyme restriction?

A

5’ Junk ,Restriction Enzyme, gene we want minus the beginning methionine 3’

165
Q

What is the order of information added to make the reverse primer using enzyme restriction?

A

5’ Junk, restriction enzyme, end of gene with stop codon (will be in reverse) 3’

166
Q

Which method of restriction enzyme cloning is not efficient, non-directional, has high rates of self-ligation and is time consuming?

A

Blunt ended cloning

167
Q

Which method of restriction enzyme cloning requires restriction digestion of insert and vector (RE should be absent internally in the insert), has two different sticky ends, directional insertion, high ligation efficiency, but is time consuming?

A

sticky ended cloning

168
Q

in oligo design the tail should have what beyond the restriction site for the enzyme to grab onto?

A

5 to 8 bp

169
Q

The reverse primer may need to encode what?

A

a stop codon

170
Q

NdeI and Ncol encode what in their restriction sites?

A

ATG

171
Q

What are the mechanisms of DNA transfer?

A

Conjugation
Transduction
Transformation

172
Q

What is conjugation?

A

The physical interaction between cells with a direct transfer of DNA from one bacterium to another

173
Q

What is transduction?

A

Virus mediated transfer of DNA between bacteria

174
Q

What is transformation?

A

When DNA is taken up from the environment by bacteria

175
Q

What are bacterial cells that are able to take up DNA from the environment called?

A

Competent cells

176
Q

Why do competent cells carry?

A

Genes that encode proteins called competence factors

177
Q

The process of making a cell competent usually involves what?

A

Ca++ that shields the negative charge of DNA

178
Q

Top10 competent E.coli cells do not contain what?

A

T7 RNA polymerase

179
Q

When you are ready to perform an expression experiment you should transform your construct into what?

A

BL21 Star (DE3) E. Coli

180
Q

Why do we use a restriction site in the insert during Top10 cloning?

A

To determine if the gene of interest is present. An additional strand will be present because of an additional cut.

181
Q

During plasmid purification, what happens to the genomic DNA and RNA?

A

DNA is excluded because it is larger and more supercooled, RNA is degraded

182
Q

What are the basic steps of Plasmid DNA purification?

A
  1. Pellet the cells
  2. Resuspend in a neutral buffer containing RNAse
  3. Lyse the cells
  4. Denature proteins and genomic DNA
  5. Capture plasmid DNA
  6. Wash t remove excess salt
  7. Elute plasmid DNA
183
Q

An alkaline solution is used to do what to cells?

A

Lyse

184
Q

What components are included in the lyse solution?

A 
SDS detergent (a detergent)
Sodium Hydroxide (a base)
185
Q

Why is a basic pH used for the lyse step?

A

It denatures the dsDNA into single strands

186
Q

What does SDS do?

A

Denature proteins

187
Q

How is the solution neutralized during plasmid purification?

A

Adding sodium acetate pH 5.5 that includes chaotropes which the proteins. As the pH drops plasmid DNA will renature due to its small and circular nature.

188
Q

Why do we not use vortexing?

A

To prevent the shearing of genomic DNA

189
Q

What happens to the DNA and proteins during the neutralizing step of plasmid purification?

A

They clump together and fall out of solution

190
Q

How do we capture the plasmid DNA in plasmid purification?

A

Option 1 ethanol precipitation
Option 2 bind the DNA to a Silva membrane - binds DNA at optimal pH and salt concentration - this can be washed with ethanol

191
Q

Why do we wash the silica membrane with ethanol?

A

The high salt is removed by the 70% ethanol wash, while DNA is bound to the membrane

192
Q

How is the plasmid eluded off the silica membrane

A

Using water or Tris buffer with no salt by applying it directly to the membrane followed by a short incubation spin

193
Q

How can the purity of the DNA be confirmed?

A

Ratio of 260/280 absorption’s

194
Q

What are the steps for agarose gel electrophoresis?

A
  1. Load sample
  2. Application of electrical field (DC)
  3. DNA separation
  4. UV transillumination and documentation
195
Q

Minipreps produce what kind of DNA?

A

Supercoiled DNA with trace amounts of nicked DNA

196
Q

What does BL21 Star (DE3) have that promotes expression?

A

Lambda DE3 lysogen which carries the gene for T7 RNA polymerase under the control of the lacUV5 promoter

197
Q

What is required to induce the expression of the T7 RNA polymerase?

A

IPTG

198
Q

BL21 Star (DE3) carries a mutated Rne gene which does what?

A

Encodes a truncated RNAse E enzyme that lacks the ability to degrade mRNA resulting in increased mRNA stability

199
Q

Translation will only start at what codon?

A

AUG/ATG

200
Q

What program do we use to find the pI or translate the gene?

A

Expasy

201
Q

BL21 Star does not contain the Ion protease and has a mutation in the outer membrane protease, Omp T. Why?

A

Reduces degradation of heterologous proteins expressed.

202
Q

Neutralizing the pH contains sodium acetate and guanidium chloride that do what?

A

Denatures proteins

203
Q

What are two types of sources for proteins?

A

Natural

  • organism
  • tissue
  • growth stage

Recombinant

  • gene sequence
  • design primers, PCR
  • clone (vector choice)
  • host cell
  • express
  • purify
204
Q

What are the molecular characteristics of your protein?

A 
Charge
Hydrophobicity
Solubility
Stability
Size
Ligand
Structural Integrity
205
Q

What two types of expression are there?

A

Intracellular and extracellular

206
Q

Is our protein soluble in cytoplasm?

A

Yes

207
Q

What are ways to lyse cells without damaging the protein?

A 
Mechanical disruption - blender
French Press
Sonic action
Freeze Thaw Cycles
Enzymatic
208
Q

How does lysozyme work?

A

Cleaves the peptidoglycan bonds and degrades the cell wall

209
Q

What are detergents?

A

Compounds with hydrophobic and hydrophilic ends, they will integrate into membranes and dis-integrate the membrane. Useful for gram-negative bacteria since they have an outer membrane over the thin layer of cell wall

210
Q

Why do we add PMSF?

A

Phenylmethylsulfonylfluoride inhibits protease

211
Q

What temperatures do you want to work at?

A

Lower 4 C

212
Q

Can you protect your protein from degradation by using special expression cells?

A

Yes

213
Q

What do you centrifuge for protein purification at?

A

30,000 G for 30 minutes to an hour

214
Q

The concentration of any salt necessary to cause precipitation of a particular protein is related to what?

A

The number and distribution of charges and of hydrophobic residues exposed.

215
Q

As salt concentration increases, protein solubility does what?

A

Decrease

216
Q

Middle stages of purification take advantage of what?

A

Properties of the protein

217
Q

What is based on the surface hydrophobic amino acids’ components of proteins?

A

Hydrophobic Interaction Chromatography

218
Q

Ion-Exchange chromatography takes advantage of what?

A

The pI of your protein

219
Q

Affinity chromatography takes advantage of what?

A

Resins contains ligand or substrate covalently attached and is used with protein affinity tag (His-tag)

220
Q

Where do you get the gene sequence from?

A

NCBI

221
Q

How do you translate it to protein (the gene)?

A

Expasy

222
Q

To find the pI of your protein what do you add to your gene?

A

V5 and his-take

223
Q

What methods are used to capture proteins that have an affinity tag?

A

Affinity chromatography

224
Q

What methods are used to capture proteins without an affinity tag?

A

Ion exchange and HIC (hydrophobic interaction chromatography)

225
Q

What is an intermediate step in protein purification with an affinity tag?

A

Ion exchange chromatography

226
Q

What is an intermediate step for protein purification without an affinity tag?

A

HIC or IEX

227
Q

What method is used to polish the protein whether it has an affinity tag or not?

A

Gel Filtration

228
Q

After reaching what optical density, the protein was expressed by the addition of 1 mM IPTG

A

OD600 of ~0.6

229
Q

What is IPTG?

A

A lactose analog

230
Q

Histidine tag coordinates with metals like what?

A

Ni and Cu

231
Q

What is used to elute a protein from an affinity column?

A

Imidazole

232
Q

What else can be used to elute a protein from an affinity column?

A

Engineered protease site

233
Q

What are other names of affinity chromatography involving metals?

A

Metal chelation chromatography or immobilized metal affinity chromatography (IMAC)

234
Q

In IMAC what are the resins made with?

A

Nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA)

235
Q

What is the pKa of his?

A

~6.5

236
Q

What is the role of the low concentration of imidazole in the lysis and wash buffers?

A

To elute out proteins with a few histidine

237
Q

What are some polishing methods?

A

IEX, GF, ultra-filtration/centrifugal concentrators

238
Q

In dialysis smaller molecules can move in and out until equilibrium is reached but it is important that what is selected?

A

The right size of membrane

239
Q

What labs were performed for AdhP expression and purification?

A

Cloned into pET101D vector with 6xhis tag
Transformed into IshotToP10 plasmid prep
Plasmid was analyzed by RE digestion
Transformed into BL21DE3
Expressed by addition of IPTG
Harvested Cells lyse and purified via Ni-NTA affinity chromatography

240
Q

What is the pH of the running buffer?

A

8.3

241
Q

What is the percentage and pH of the stacking gel?

A

4% pH 6.8

242
Q

What is the percentage and pH of the resolving gel?

A

7 to 15% pH 8.8

243
Q

SDS Page Theory involves what?

A

The movement of charge particles under the influence of an electric field

244
Q

Negatively charge molecules move toward what?

A

The positive anode

245
Q

Positively charged molecules move toward what?

A

The negatively charged cathode

246
Q

What is the reaction for anode (oxidation)?

A

H20 -> 2 H + 1/2 O2 + 2 e

247
Q

What is the reaction for cathode (reduction)?

A

2e + 2H2O -> 2 OH + H2

248
Q

How many more bubbles arise from the cathode?

A

Twice as many

249
Q

The pH does what in the cathode chamber?

A

Increases

250
Q

The pH does what in the anode chamber

A

Decreases

251
Q

Acrylamide is a dangerous what?

A

Neurotoxin

252
Q

What is TEMED used for?

A

Helps cleave persulfate

253
Q

The free radical (sulfate radical) does what?

A

Forms crosslinkages between N-methylenebisacrylamide (monomer)

254
Q

Acrylamide solutions come in what ratios?

A

19:1, 29:1, 37.5:1

Ex for every 19 acrylamide there is 1 bisacrylamide

255
Q

SDS coats to a density of what?

A

1.4g SDS per g polypeptide

256
Q

What is SDS used for?

A

Denatures protein and gives it a negative charge (protein is effected by differences in size only)

257
Q

Reducing agents like dithiothreitol (DTT) do what?

A

Break covalent disulfide bonds (cystines) and converts into monomers

258
Q

what happens in the stacking gel?

A

Proteins are concentrated as a thin band at the interface

259
Q

What happens in the resolving gel?

A

Proteins get separated based on size

260
Q

What should the properties of the loading dye be?

A

Viscous
Color to track run
SDS and reducing agent
PH of 6.8

261
Q

Who developed the SDS-Page?

A

Ulrich Laemmli

262
Q

SDS-Page is called discontinuous electrophoresis because of what?

A

The pH differences from 8.3 to 6.8 to 8.8

263
Q

What charge does the protein have in the running buffer?

A

Negative

264
Q

What charge do the proteins have in the stacking gel?

A

Neutral

265
Q

What charge do the proteins have in the resolving gel?

A

Negative

266
Q

The charge distribution is based off what amino acid?

A

Glycine

267
Q

The fast moving Cl- ions and mostly neutral glycine molecules create what?

A

A zone of low conductance or high resistance

268
Q

At constant current V=IR, the high resistance leads to what?

A

A localized increase in the electric field

269
Q

For SDS mobility is directly proportional to what?

A

The charge of the molecule and is inversely proportional to the frictional coefficient

270
Q

When denatured by heat and coated with SDS, what becomes nearly the same for all polypeptides?

A

Shape and charge

271
Q

Coomassie Brillant blue R 250 (different from dye used in Bradford assay G-250) has what charge so that it will bind to the protein?

A

Positive

272
Q

What is the equation for relative mobility?

A

Rm = distance migrated by protein/distance migrated by the dye front

273
Q

How can you determine the MW of an unknown sample

A

Plotting log of known molecular weights with relative mobility

274
Q

One unit of enzyme is defined how?

A

The amount of enzyme required to generate 1 micromole of product in one minute at a given pH and temperature (Standard 25 C or 37 C)

275
Q

Activity coeffiecient for NAD+ at 260 is what?

A

18,000

276
Q

Activity coefficient for NADH at 260 is what?

A

15,000

277
Q

Activity coefficient for NADH at 339?

A

6,220

278
Q

Activity coeffiecient for NAD+ at 339 N.M.?

A

0

279
Q

What does AdhP do?

A

Catalyzes the oxidation of alcohol to aldehyde in a zinc dependent manner in the presence of cofactors NAD+ and NAD+ get reduced to NADH (absorbs at 340)

280
Q

Activity is express how?

A

As micromoles of product formed per min

281
Q

How do you determine total activity?

A

Units*ml

282
Q

How do determine specific activity?

A

Divide the activity (units) by protein (mg)

283
Q

How to you determine the fold of purification?

A

Divide the specific activity by the specific activity of the Lysate

284
Q

How do you determine % yield?

A

(Total activity/total activity of lysate)* 100

285
Q

In IEF (isoelectric focusing) Gel the proteins stop moving when what?

A

The net charge of the protein is zero.

286
Q

Isoelectric focusing separates proteins based on what?

A

Their pI

287
Q

What is 2D electrophoresis?

A

Separates first by pI and second based on size

288
Q

What technique is used to detect the presence of a specific protein in a mixture to confirm the identity of a particular protein?

A

Western Blot

289
Q

What does the Western blot take advantage of?

A

The highly specific interaction of antigen to an antibody

290
Q

What is an antigen?

A

A molecule that can induce an immune response

291
Q

What is an antibody?

A

A Protein produced by specialized white blood cells to recognize and inactivate an antigen

292
Q

What is an epitope?

A

A portion of an antigen that is recognized by the antibody

293
Q

What are steps of Western Blot?

A

Separate on SDS page
Transferred to a positively charged membrane
Bound by antibodies
Detected by reaction - the interaction between the antigen and antibody is detected by a colorimetric or fluorescent reaction

294
Q

Western Blot overview steps are what?

A 
SDS-Page
Transfer
Block
Rinse
Antibody
Rinse
Color Development
295
Q

What is the difference between nitrocellulose membranes and polyvinylidene difluoride membranes?

A

Nitrocellulose are excellent protein binding and retention capabilities but are brittle and not used when proteins need to be stored

(PVDF) have superior mechanical strength making it suitable for stripping/reproving and for further characterization techniques such as sequencing and proteolytic. Take care when staining

296
Q

What is the fragment antigen binding?

A

A region on an antibody that binds to antigens

297
Q

The fragment constant (Fc) composition will be what for all antibodies produced in an organism?

A

Identical

298
Q

What is the difference between monoclonal and polyclonal antibodies?

A

Monoclonal
Specificity for a single epitope
Less sensitive since only a single antibody molecule binds to each target

Polyclonal
Varying specificities to multiple epitopes
More sensitive

299
Q

There are how many antibodies involved in Western blotting?

A

Two , primary, secondary - enzyme conjugated

300
Q

In our experiments the primary antibody is directly conjugated to what?

A

The HRP enzyme

301
Q

What is a mouse monoclonal IgG2a antibody that is itself conjugated to an enzyme?

A

Anti-V5-HRP the enzyme is horse radish peroxidase (HRP)

302
Q

A chromogenic substrate is added that HRP will catalyze to produce what?

A

Color

303
Q

HRP is a heme containing oxidoreductases enzyme that catalyzes the oxidation of 4-Chloro-I-naphthol (4CN) in the presence of what?

A

Hydrogen peroxide

304
Q

what is the product formed by HRP?

A

An insoluble purple quinone

305
Q

When you want to clone a gene into a TA vector, what DNA polymerase should you use in the PCR and why?

A

Taq Polymerase it adds a A at the end of the 3’ end of the PCR product so that it can be inserted directionally into a TA vector.

306
Q

When you want to clone a gene into a TOPO vector, what DNA polymerase should you use in the PCR and why?

A

DNA topoisomerase because it will insert the PCR product in the correct direction

307
Q

What is the T7 promoter and T7 terminator sequence

A

T7 promoter, lacO, Xba 1, RBS, Topoisomerase polymerase sites, Sac I, BstB I, V5 epitope, 6xHis, Stop,T7 terminal

Biochemical Methods (5 decks)

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