ELECTROPHORESIS Flashcards
(118 cards)
1
Q
is the movement of molecules by (DNA or RNA) by (applying specific voltage) an electric current
A
Electrophoresis
2
Q
This can occur in air or solution or in a matrix to limit migration and contain the migrating material
A
Electrophoresis
3
Q
True or False
Electrophoresis is commonly applied to the analysis of nucleic acids and proteins molecules
A
T
4
Q
True or False
Nucleic Acids are mostly positive charged charged
A
F
5
Q
Each phosphate group on a nucleic acid polymer is ________ making the molecule negatively charged
A
ionized
6
Q
Under an electric current, DNA/RNA will migrate toward the?
A
positive pole (anode)
7
Q
In a matrix of agarose or polyacrylamide, migration under the pull of the current is impeded, it depends on the? (2)
A
size of the molecules
the spaces in the gel matrix
8
Q
Things to note of Nucleic Acids in Electrophoresis (3)
A
It is affected by the size and the charge of the
particle
The nucleic acid will move toward the positive pole
(anode) because nucleic acids are negatively
charged.
From cathode it will migrate to anode
9
Q
When phosphate group is of course ionized, it will be (1)___________ charged, so that would confer a (2) __________to your DNA and RNA.
A
- Positively
- Negativity
10
Q
provide resistance to the movement of molecules under the force of an electric current
A
THE GEL SYSTEM
11
Q
Gel system prevent diffusion and reduce convection
currents so that the separated molecules form
a defined group, or called as?
A
band
12
Q
can then serve as a support medium for analysis of
the separated components
A
gel
13
Q
The best gel matrix would be? (3)
A
unaffected by electrophoresis
simple to prepare
amenable to modification.
14
Q
The 2 best gel matrix in the analysis of NA in Electrophoresis
A
Agarose and Polyacrylamide
15
Q
Agarose Gel
The size of the DNA is affected by its?
A
concentration
16
Q
Agarose Gel
High concentration : ?
Low concentration : ?
A
impede migration
weak gel
17
Q
Small pieces of DNA (50 to 500 base pairs [bp]) are resolved
on?
A
Higher agarose concentrations (ex. 2% to 3%)
18
Q
Larger fragments of DNA (2,000 to 50,000) are best resolved
in?
A
lower agarose concentrations (ex. 0.5% to 1%)
19
Q
The gel strength of any concentration of agarose will also? (2)
A
decrease over time
with exposure to chaotropic agents such as urea.
20
Q
DRAWBACK of Agarose gel Concentrations?
A
Agarose concentrations above 5% and below 0.5% are not practical.
21
Q
Identify what type of Electrophoresis.
-consists of very large pieces (50,00 to 250,000 + bp) of DNA
-Used Bacterial typing for epidemiological purposes
A
Pulse-field Gel Electrophoresis
22
Q
Pulse-field Gel Electrophoresis.
For these very large DNA molecules, pulses of current applied to the gel in what dimension?
A
alternating dimensions to enhance migration
23
Q
The four approaches of Pulse-field Gel Electrophoresis.
A
FIGE
TAFE
RGE
CHEF
24
Q
The simplest approach to PFGE is?
A
field-inversion gel electrophoresis (FIGE) .
25
What approach is this in PFGE?
-works by alternating the positive and negative
electrodes during electrophoresis
-
In this type of separation, the DNA goes
periodically forward and backward
field-inversion gel electrophoresis (FIGE) .
26
is used for applications that require the resolution of chromosome-sized fragments of DNA, such as in bacterial typing for epidemiological purposes.
Alternating-field electrophoresis
27
will yield a set of fragments that produce a band pattern specific to each type of organism. By comparing band patterns, the similarity of organisms isolated from various sources can be assessed. This information is especially useful in determining the epidemiology of infectious diseases.
Enzymatic digestion of genomic DNA
28
- Require a catalysts
- Higher resolution capability for smaller
fragments
Polyacrylamide Gels
29
catalysts of Polyacrylamide Gels (3)
1. ammonium persulfate (APS)
2. N,N,N′,N′-tetramethylethylenediamine (TEMED)
or 3. light activation
30
Acrylamide, in combination with the cross-linker ___________________, polymerizes into a matrix that has consistent resolution characteristics
methylene bisacrylamide
31
____________ is a natural polymer from living organisms?
_____________ is a synthetic material
agarose
polyacrylamide
32
This gel allows precise control of the polymer properties and higher resolution.
polyacrylamide
33
how does agarose gel polymerize?
cooling
34
how does polyacrylamide gel polymerize?
polymerizations requiring a catalyst (APS + TEMED/Light Activation)
35
_________ produces free oxygen radicals in the presence of TEMED to drive the polymerization mechanism
APS
36
What catalyst is this?
Free radicals are generated by a photochemical process using riboflavin plus TEMED.
Light activation
37
If Light activation is used:
Free radicals are generated by a photochemical process using?
riboflavin + TEMED
38
If light activation is used, Excess oxygen inhibits the polymerization process.
Therefore ________or ____________ of the gel solution is done before the addition of the nucleating agents.
de-aeration or the removal of air
39
The main advantage of polyacrylamide over
agarose is??
higher resolution capability of polyacrylamide for small fragments.
40
With single-base resolution, polyacrylamide
gels are used for: (4)
-nucleic acid sequencing
-mutation analyses
-nuclease protection assays
-other applications requiring high resolution of nucleic acids
41
What type of electrophoresis?
-separates particles by size and charge
-Size and charge (charge/mass ratio)
-Increased sensitivity and immediate detection
CAPILLARY ELECTROPHORESIS
42
CAPILLARY ELECTROPHORESIS
size (small, ____migration; large, ____migration)
charge (________, fast migration; ________, slow migration)
size (small, fast migration; large, slow migration)
charge (negative, fast migration; positive, slow migration)
43
CAPILLARY ELECTROPHORESIS
Because the size and charge of DNA work counter to each other, a __________ in the capillary will resolve the DNA fragments mostly according to size.
polymer (gel)
-so bale more on SIZE ang capillary electrophoresis.
44
CAPILLARY ELECTROPHORESIS
Negatively charged molecules are completely (1) _________ at ______pH, whereas positively charged solutes are completely
(2)_________ in ____-pH buffers
ionized, high
protonated, low
45
True or False
In Capillary Electrophoresis, Nucleic acids do not separate well in solution.
T
(As the size or length of a nucleic acid increases (slowing migration), so does its negative charge (speeding migration),
effectively confounding the charge/mass resolution
46
In Capillary Electrophoresis, It is VERY important that the nucleic acids are??
completely denatured (single stranded) so that it will be
separated according to its size because the secondary
structure (dsDNA) will affect the migration speed.
47
Compared with traditional slab gel electrophoresis, the
capillary system has the advantages of?
Increased sensitivity and immediate detection
48
to carry the current and protect the samples during electrophoresis
BUFFER SYSTEM
49
Tris-buffers
Most common NA?
DNA
50
Types of Tris-Buffers (3)
1. Tris-borate EDTA (TBE)
2. Tris phosphate EDTA (TPE)
3. Tris acetate EDTA (TAE)
51
Basaha ra (Tris-Buffers)
TBE has a greater buffering capacity than TAE. Although the
ion species in TAE are more easily exhausted during
extended or high-voltage electrophoresis, DNA will migrate
twice as fast in TAE than in TBE in a constant current.
52
modify sample molecules in ways that affect their migration
Buffer Additives
53
Examples of these buffer additives are: (3)
1. Formamide
2. Urea
3. Detergents
54
Denaturing agents that break hydrogen bonds between complementary strands or within the same strand of DNA or RNA (2)
(Formamide & Urea)
55
___________ and heat added to DNA and RNA break and
block the hydrogen-bonding sites, hindering complementary sequences from reannealing. As a result, the molecules become long, straight, unpaired chains.
Formamide
note. block the hydrogen-bonding sites, no anneal, and long, straight, unpaired chains
56
________ and heat in the gel systems maintain this conformation such that intrachain hybridization (folding) of the nucleic acid molecules does not affect migration speeds, and separation occurs strictly according to the size or length of the molecule.
Urea
note. intrachain hybridization (folding) and strict separation to size and length
57
RNA Denaturing Agents that reacts with amino groups on the RNA to prevent base pairing between complementary nucleotides and with aldehydes (e.g., formaldehyde, glyoxal) which also disrupt base pairing.
methylmercuric hydroxide (MMH)
58
ELECTROPHORESIS EQUIPMENT
-are run in acrylic gel boxes or baths that are divided into two parts, with a platform in the middle on which the gel rests
Horizontal gels
59
ELECTROPHORESIS EQUIPMENT
What colored connector is attached to positive port? (1)
What colored connector is attached to negative port? (2)
RED
BLACK
60
ELECTROPHORESIS EQUIPMENT
NA will migrate toward the?
Positive (red) pole
61
ELECTROPHORESIS EQUIPMENT
-placed across the box at each end of the bath compartments are connected to a power supply through the walls of the container
The electrodes (platinum wires)
62
ELECTROPHORESIS EQUIPMENT
-is submerged in electrophoresis buffer that fills both compartments and makes a continuous system through which the current flows.
Gel
63
ELECTROPHORESIS EQUIPMENT
The THICKNESS OF THE GEL and THE VOLUME OF THE BUFFER affect the _________, and therefore the migration of the sample, so these parameters are kept ________ for consistent results.
current
constant
64
ELECTROPHORESIS EQUIPMENT
Because the gel is submerged throughout the loading and electrophoresis process, horizontal gels are sometimes referred to as ??
submarine gels
65
The volume of the gel solution will determine the?
thickness of the gel.
66
True or False
Gel is mixed with agarose and Polyacrylamide
F
Gel is mixed with buffer
67
Molten agarose is cooled to between what temp?
55C and 65C
68
inserted into the top of the gel to create holes, or wells, in the gel into which the sample will be loaded
Comb
69
Comb
**Size of the teeth = ?**
**# of the teeth = ?**
capacity
of wells
70
Two types of combs used in polyacrylamide electrophoresis
1. Regular combs- horizontal gel
2. Sharkstooth combs - vertical gel
71
Combs
________ gels are cast between glass plates that are separated by spacers.
vertical gel (Sharkstooth combs)
72
Combs
The spacers determine the thickness of the gel, ranging from _____ to ______mm.
0.05 to 4 mm.
73
Combs
The bottom of the gel is secured by _____or by a ______ in specially designed gel casting trays.
tape or by a gasket
74
Combs
After the addition of catalyst and nucleating agents, the (1) ___________ is poured or forced between the glass plates with a (2) _______ or a (3) _______
1 Liquid acrylamide
2 Pipet
3 Syringe
75
Combs
For light-activated polymerization, the gel between the glass plates is exposed to a _________
light source.
76
Comb
It is important to prevent air from getting into the gel or beneath the comb. ________ will form discontinuities in the gel, and oxygen will inhibit the polymerization of the acrylamide.
Bubbles
77
The comb is of a thickness equal to that of the
________ so that the gel will be the same
thickness throughout.
spacers
78
True or False
Combs are only used in Polyacrylamide Gel Electrophoresis and not on Agarose
F
79
Combs
for vertical gels/Sharkstooth combs, its usually ____________
horizontal/Regular combs, could either be _________ or _________
polyacrylamide
agarose or polyacrylamide gels
80
Gel Loading consists of (2)
Tracking dye
Density agent
81
used to monitor the progress of the electrophoresis run
Tracking dye
82
The dyes migrate at specific speeds in a given gel concentration and usually run ahead of the SMALLEST fragments of DNA
Tracking dye
83
3 ex. of Density Agents
Ficoll
sucrose
glycerol
84
increases the density of the sample as compared with the electrophoresis buffer.
Density Agents
85
DAWBI?
kamo nay memo sa number2 sa tracking dye uy kapoy
86
Two Most Common Tracking Dye
Bromophenol blue and Xylene cyanol green
87
is a tracking dye that is used for many applications
Bromophenol blue
88
is another of the chromophores used as tracking dyes for both agarose and polyacrylamide gels
Xylene cyanol green
89
True or False
Tracking dyes are associated with the sample DNA, and thus they affect the separation.
F
Tracking dyes are not associated with the sample DNA, and thus they do not affect the separation.
90
DETECTING SYSTEM (2)
Fluorescent Dyes
Silver Stain
91
The agents used most frequently for visualization of bands
after electrophoresis are (2)
Fluorescent Dyes
Silver Stain
92
an intercalating agent and most widely used dye in EARLY dna and rna analyses.
Ethidium bromide
93
EtBr Under excitattion with UV light (nm)?
300 nm
94
EtBr in DNA emits visible light at (nm)?
590 nm
95
DNA separated in agarose or acrylamide and exposed to EtBr will emit ______ light when illuminated at "300 nm"
orange
96
After electrophoresis, the agarose or acrylamide gel is soaked in a solution of ____ to _____-mg/mL EtBr in running buffer (TAE, TBE, or TPE) or in TE.
0.1- to 1-mg/mL EtBr
97
ETBR
After soaking or running in dye, the DNA illuminated with UV light (300nm) will appear as ________ bands in the gel.
orange
98
Minor Groove-Binding Dyes (2)
SYBR green
SYBR gold
99
SYBR green I - ?
SYBR green II - ?
SYBR green I - dsDNA
SYBR green II - ssDNA, RNA
100
SYBR green I differs from EtBr in that it?
it does not intercalate between bases; it sits in the minor groove of the double helix.
101
SYBR green in association with DNA or RNA also emits (visible) light in the orange range (nm)?
522 nm
102
In agarose gel electrophoresis, SYBR staining is __ to ___ times more sensitive than EtBr
25 to 100
so mas SENSITIVE si SYBR kaysas Etibromide nato
103
Fluorescent Dyes
_________ can also be added directly to the DNA sample
before electrophoresis
SYBR green
so SYBR ma add na b4 electrophoresis unlike sato ETBR nga after pa kay mag soak2 paman nasya
104
but in SYBR, basaha lang
DNA prestaining decreases the amount of dye required for
DNA visualization but lowers the sensitivity of detection and
may, at higher DNA concentrations, interfere with DNA
migration through the gel.
105
True or False
Because SYBR green is not an intercalating agent, it is mutagenic
F
Because SYBR green is not an intercalating agent, it is not as mutagenic
106
True or False
SYBR green is safer to use than EtBr
T
107
True or False
Ethidium bromide is good for Real time PCR
F
SYBR green
108
What is this type of fluorescence dye?
-fluorescence increases more than 1,000- fold upon binding to double- or single-stranded DNA or to RNA
SYBR Gold
109
Like SYBR green, SYBR gold is excited by UV
light (nm)?
300 nm
110
SYBR gold emits (visible) light at (nm)?
537 nm
111
Another sensitive staining system originally developed for protein visualization, increased sensitivity.
Silver Stain
112
Silver Stain consists of 2 procedures?
Silver diamine (ammoniacal silver)
Silver nitrate
113
Silver Stain
After electrophoresis, the sample is fixed with (2)
methanol
acetic acid
114
Silver Stain
The gel is then impregnated with ammoniacal
silver (silver diamine) solutions or silver nitrate in a ___________ solution.
weakly acid
115
Silver Stain
Interaction of silver ions with acidic or nucleophilic groups on the target results in _________ or ______________ of metallic silver under optimal pH conditions.
crystallization or deposition of metallic silver
116
Silver Stain
The insoluble black silver salt precipitates upon
introduction of ________ in a weak acid solution, or alkaline solution for ____________
formaldehyde (weak acid sol'n)
silver nitrate (alkaline sol'n)
117
Silver Stain
____________ is best for thick gels
____________ is considered to be more stable
silver diamine
silver nitrate
118
These are especially useful for protein analysis
and for detection of limiting amounts of product.
Silver Staining
