LESSON 9 GEL ELECTROPHORESIS Flashcards
1
Q
Technique used for the separation, detection, visualization and sometimes purification of biomolecules DNA, RNA and protein using an electric field applied to a gel matrix
A
gel electrophoresis?
2
Q
A technique used to separate DNA fragments according to their size.
A
gel electrophoresis?
3
Q
Purpose gel electrophoresis It is used to visualize, measure the size of the DNA fragments and even identify contaminants from the
[?]: millions of DNA produced
[?]: to check if you will be “pasting” the correct gene
A
- PCR test
- DNA cloning/ Recombinant technology
4
Q
Sample has already run thru the electric current and have migrated from the
A
negatively charged(cathode) to the positively charged (anode) area
5
Q
: diabetic patients (9 patients)
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TGF-B gene
6
Q
o standard/reagent/DNA ladder
A
M (molecular weight markers)
7
Q
o serves as a basis for the length of DNA with a unit of “bp” (base pair)
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M (molecular weight markers)
8
Q
= 1 kbp
A
1,000 bp
9
Q
: DNA ladder
A
Lane 1
10
Q
: has a DNA fragment of 1,500 bp
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Lane 2
11
Q
: has a DNA fragment of 600 bp (with TGF-B gene)
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Lane 3-9
12
Q
: fluorescent dye stain
A
Ethidium bromide
13
Q
Used for visualization under UV light/transilluminator
A
Ethidium bromide
14
Q
To get the fragment (?), elute or cut the agarose gel (DNA portion) → wash
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600 bp
15
Q
acts as a strainer for the separation of particles
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gel in electrophoresis
16
Q
: fastest to migrate
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Small DNA/RNA fragemnts/shorter base pairs
17
Q
voltages involved
A
(positive and negative charges)
18
Q
separation according to
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size of bp
19
Q
2 kbp from 10 bp =
A
2 kbp gene
20
Q
DNA fragments are [?] (loaded at the cathode), so they move towards the positive electrode (anode).
A
negatively charged
21
Q
presence of phosphate group (phosphoric acid)
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negatively charged
22
Q
Separation takes
A
30-45 mins
23
Q
: DNA fragments will overpass
A
Long electrical runs
24
Q
Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel [?] than large ones.
A
faster
25
Voltage:
Depends on the protocol
60-120 V
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Separated in [?]; test is optimized; proper migration to properly identify their sizes
bands
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REAGENTS EQUIPMENTS AND REAGENTS/ELECTROPHORESIS SYSTEM
Power supply and chamber
Gel casting tray/gel box/ gel chamber:
Gel comb:
Buffer dam or tapes:
28
a source of power supply
Power supply and chamber
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: hold/mold the agarose
Gel casting tray/gel box/ gel chamber
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: create holes or sample wells in the gel where the samples are loaded
Gel comb
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: limits the movement of the agarose gel
Buffer dam or tapes
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Matrix where samples are loaded and DNA migrates
Agarose
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Commonly used in gel electrophoresis
Agarose
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For big DNA fragments from 50-20,000bp
Agarose
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High resolving power for short 5-500bp
Polyacrylamide
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Horizontal
Agarose
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Vertical
Polyacrylamide
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High agarose conc: smaller pore size
Agarose
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Routinely 6-15%
Polyacrylamide
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Set as it cools
Agarose
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Sets by chemical reaction once crosslinking occurs
Polyacrylamide
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Polysaccharide extracted from sea weed
Agarose
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Crosslinked polymer of acrylamide
-Neurotoxin
-P.A.: nontoxic
Polyacrylamide
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Easy to prepare
Agarose
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Sets quickly
Agarose
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Can be refrigerated
Agarose
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Non toxic
Agarose
48
Pore size can be manipulated
Agarose
49
Buffers can alter the resolution and run times
Agarose
50
Large range of DNA can be separated
Agarose
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Produce reproducible and consistent results
Polyacrylamide
52
Pores uniform in size (smaller)
Polyacrylamide
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Non-reactive/inert with samples
Polyacrylamide
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Very high resolving power -clear DNA fragments
Polyacrylamide
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Pore size can be manipulated
Polyacrylamide
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Buffers can alter the resolution and run times
Polyacrylamide
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Do not have a uniform pore size
Agarose
58
Low percentage gels: may be weak and may break
Agarose
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High percentage gels: brittle and may not set evenly
Agarose
60
Potent neurotoxin (Acrylamide)
Polyacrylamide
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PPE must be used though
Polyacrylamide
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PA is non-toxic
Polyacrylamide
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Bubble may form in pouring -due to chem rx
Polyacrylamide
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Longer wait time
Polyacrylamide
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Based on the length of plasmid/bp ; ↓agarose conc = weaker ; Closest to the lower limit
Agarose Gel Conc (1-2%)
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: Large pores size; may be weak and may break
Low percentage gels
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: Small pore size; brittle and may not set evenly
High percentage gels
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- the larger fragments are much better resolved in the [?] gel
0.7%
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- the small fragments separated best in [?] agarose (better separation)
1.5%
70
- the [?] fragment i indicated in each lane
1000 bp
71
are sufficiently pure to avoid problems such as electroendosmosis
- Agarose preparations
72
a solvent flow toward one of the electrodes, usually the cathode (negative), in opposition to the DNA or RNA migration,
electroendosmosis
73
slows and distorts the migration of the samples, reducing resolution and smearing the band
electroendosmosis
74
(prevented by choosing the correct agarose conc)
electroendosmosis
75
QUESTION: You wish to perform an electrophoretic resolution of your restriction enzyme-digested DNA. The sizes of the expected fragments range from 100 to 500 bp. You discover two agarose gels polymerizing on the bench. One is 0.5% agarose; the other is 2% agarose. Which one might you use to resolve your fragments?
2%: used for shorter DNA fragements
76
Solution that will conduct electricity because of their hydrogen ion concentration and maintain the pH
Buffers
77
Gives the charge on the agarose gel to allow flow of elec to the gel matrix
Buffers
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: chelator of nucleases
EDTA
79
cancels out enzymes that coud disrupt DNA against DNAses
EDTA
80
Low buffering capacity
tris-acetate-EDTA (TAE)
81
High buffering capacity (does not change the pH)
tris-borate-EDTA (TBE)
82
Fast DNA migration (better conductivity)
tris-acetate-EDTA (TAE)
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Slow DNA migration
tris-borate-EDTA (TBE)
84
Overheats and exhausted during extended/ repeated electrophoresis
tris-acetate-EDTA (TAE)
85
Adv: Won't overheat quickly during long runs (controls temp to prevent melting)
tris-borate-EDTA (TBE)
86
Good for large fragments (>2kb)
tris-acetate-EDTA (TAE)
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For smaller fragments (<2kb)
tris-borate-EDTA (TBE)
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Non toxic
tris-acetate-EDTA (TAE)
89
Carcinogenic
tris-borate-EDTA (TBE)
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Absent Borate
tris-acetate-EDTA (TAE)
91
Borate present as enzyme inhibitor (more expensive)
tris-borate-EDTA (TBE)
92
Lower cost
tris-acetate-EDTA (TAE)
93
Higher cost
tris-borate-EDTA (TBE)
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used at 1X for the electrophoresis of RNA as the running buffer to separate RNA samples on agarose and formaldehyde-agarose (denaturing) gels
MOPS buffer (Buffer additive)
95
Adds mass/ density to the DNA sample so that it will go into the well makes it sink to the bottom
Loading dye
96
mixed w/ DNA samples and added thru the wells created from the gel comb
Loading dye
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absence: DNA could float off the gel
Loading dye
98
(sinkers: [?])
Glycerol, sucrose and ficoll
99
Adds blue color so you can see what you are pipetting
Tracking dye
100
Tracking dyes:
(Bromophenol blue – most common, Cresol Red, Orange G, tartrazine, xylene cyanol)
101
to know whether electrophoresis (EP) has started; EB can be added before starting EP
Tracking dye
102
Contains DNA fragments with known sizes that are compared to our experimental setups and are used to estimate the sizes of DNA fragments in your DNA sample
Standard/Molecular weight markers/DNA ladder
103
basis to compare the DNA set-up
Standard/Molecular weight markers/DNA ladder
104
should be performed on the same agarose gel, not separated to prevent varying temp, run, conc
DNA ladder
105
1. What lane contains the longest DNA fragment?
1
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2. What lane contains the shortest DNA fragment?
2
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3. What lane contains 1500bp DNA fragment?
3
108
RNA ladders are usually provided with a loading buffer containing a
denaturant/ denaturing agents.
109
: for single-stranded DNA/linear RNA
Denaturing agents
110
Denaturing agents
Formide
Urea/Chaotropic agents
Sodium dodecyl sulphate/SDS (thermofisher)
111
Denaturants purpose:
1. help maintain RNA in [?] (linear in shape)
2. Prevents the formation of [?] (folding)
3. Allow more predictable sample [?] results.
single-stranded form
secondary structure
migration and separation
112
When performing RNA gel electrophoresis, [?] should be avoided, because their use under denaturing conditions can lead to atypical separation patterns due to separation of the double strands.
DNA ladders
113
run under the condition that disrupts the natural/native structure of DNA/RNA or protein, which are unfolded into linear chains.
Denaturing gels
114
run under conditions that no disruption of structure is introduced to analytes.
Non-denaturing (native) gel
115
The speed of these macromolecules moving through a gel depends only on their linear length and the mass-to-charge ratio; thus, only the primary structure is analyzed.
Denaturing gels
116
In this case, the cross-sectional area of the macromolecule, in addition to the molecular mass and intrinsic charge, is also a factor for gel separation, allowing for analysis of all four levels (primary, secondary, tertiary, and quaternary levels) of the biomolecular structure.
Non-denaturing (native) gel
117
Urea is usually to denature DNA or RNA, while sodium dodecyl sulfate is used for protein denaturing
Denaturing gels
118
Fluorescent dye
Ethidium Bromide
119
May be added during the making of agarose gel or after the migration
Ethidium Bromide
120
DNAs are visualized by staining the gel
Ethidium Bromide
121
will bind to DNA and will fluoresce in UV light.
Ethidium Bromide
122
Carcinogenic
Ethidium Bromide
123
: UV lightbox to visualize the EtBr stained gel
Transilluminator
124
Safer alternative, more expensive
SYBR Green/ SYBR Safe/SYBR Gold
125
seen under UV
SYBR Green/ SYBR Safe/SYBR Gold
126
ELECTROPHORESIS PROCEDURE
127
1. Prepare agarose
[?] o Ex: 0.5 agar in 50mL buffer will result to 1%
Once the tablets have dissolved chemically by stirring, heat the solution in a microwave at full power for short bursts of [?] (prevent over boiling; as soon as bubbles appear)
Overboiling: [?] (buffer as a diluent)
Stop when you see [?]
Add [?
[?] of DNA Safe Stains to every [?] of gel solution
[?]: It emits green fluorescence when bound to dsDNA (double) and red fluorescence when bound to ssDNA (single) or RNA
Gel percentage=(grams of agarose / milliliters of buffer) × 100%
20-30 seconds
Increased percentage concentration and high ionic concentration
bubbles
ethidium bromide or DNA safe stain
1 uL ; 20 mL
DNA safe stains
128
2. Preparing the gel box for casting
Ensure the [?] are installed correctly, then install one of the [?].
Place the [?]
black buffer dams ; combs
tape
129
3. Pouring the gel
Once the agarose is fully melted and dissolved, let the solution cool to about [?]. The beaker should feel hot, but not too hot to touch.
[?]: warp/melt the casting tray and comb
Pour at [?] mark on a level surface (or 0.5 cm/5 mm)
It will take [?] to solidify at RT, one can place it into the fridge
55°C
Too hot
6-7mm
30 minutes
130
affects the DNA fragments
Thickness of the gel
131
4. Remove the comb and buffer dams/ tape
Be careful not to damage the [?] as you remove the comb and shutters. When removing the [?], make sure not to pierce the gel.
gel
comb
132
5. Gel buffer
Use your prepared 0.5x TBE buffer solution again, and pour it over the gel until the gel is fully covered. The buffer should reach about [?] above the gel.
2-3mm
133
6. Loading Dye
Before loading each sample, you need to add [?] to each sample
loading dye
134
7. Loading the gel with sample
Using a fresh pipette tip, load [?] (w/ DNA sample and loading dye) of the sample into an empty well. Discard your pipette tip, then make a note so that you remember which sample is in which well.
Repeat this for each sample, taking care to use a [?] each time.
5ul
fresh pipette tip
135
8. Run gel at constant voltage until band separation occurs
Gently close the [?] by sliding the lid onto the gel tray. Then connect the leads to the power supply. Be careful not to spill anything.
Longer electrophoretic runs will increase the [?] between fragments
Adequate separation is important for analysis of [?], especially those that are close in size.
However, if the electrophoresis is conducted for too long, DNA bands may [?] of the gel
The [?], the faster the DNA will travel through the gel. However, voltages that are too high can possibly [?] of DNA bands.
The [?] affect electrophoretic separation. For example, DNA samples will migrate faster in a 0.8% gel compared to a 1% gel. Likewise, samples will migrate faster in a 20 ml gel (6 mm thick) versus a 30 ml gel (8 mm thick) with the same 7 x 7 centimeter dimensions.
gel box
separation
DNA fragments
migrate off the end
higher the voltage ; melt the gel or cause smearing or distortion
gel concentration and volume (thickness)
136
depends on the bp being tested
Voltage
137
9. View DNA on UV light box and document results
Identify the DNA base pair based on the
DNA ladder
138
10. DNA/RNA purification from a mixture
Separate the DNAs in [?]
Cut the required [?]
Extract the DNA from gel thru [?]
Agarose gel
(DNA + Gel) band
elution or other methods
139
Requires training and precautions for radioactive safety
Radioactive stains
140
Detects or labels samples by radioactive probes or nucleotides
Radioactive stains
141
Common for detection of oligonucleotides
Radioactive stains
142
Considered most sensitive (but poses health risk)
Radioactive stains
143
Radioactive stains labels
32P; 33P; 3H labels
144
the gel is exposed to X-ray film after ectrophoresis for documentation
Autoradiography
145
The intensities of radiolabeled bands may be measured by densitometry for quantitation.
Autoradiography
146
Plasmid which is
4070bp (4kb)
147
: DNA cutting enzyme which recognize or a few target sequences and cuts DNA a-- those sequences
Ecor1
148
acts as a cutting enzyme/scissor
Ecor1
149
restriction enzyme found in E. coli
Ecor1
150
seen using eletrophoresis
Ecor1
151
1. [?] cleave DNA into smaller segments of various sizes.
2. [?]are loaded into wells in a porous gel. The gel floats in a buffer solution within a chamber between two electrodes.
3. When an electric current is passed through the chamber, DNA fragments move toward the [?].
4. Smaller DNA segments move [?] than larger DNA segments.
Restriction enzymes
DNA segments
positively-charged cathode
faster and farther
152
Cut and combine to a gene with [?] with a pasting enzyme (DNA ligase) to create a new plasmid
1,000 bp
153
Get the 1,200 bp and pair with a gene with [?] to create another recombinant DNA
2,870 bp
154
Agarose tablets/powder are placed in the beaker
1. Prepare agarose
155
Buffer/diluent: tris-borate EDTA or distilled water
1. Prepare agarose
156
2.5 uL DNA stain : 50 mL of gel solution
1. Prepare agarose
157
Comb serves as a mold for the wells
2. Preparing the gel box for casting
158
Buffer dams/tape: black
2. Preparing the gel box for casting
159
4. Remove the comb and buffer dams/ tape
Wells are created
160
Add/overlay the agarose gel w/ the gel buffer
5. Gel buffer
161
Pipette loading dyes to the sample (sinker to have higher density: glycerol, stain: bromophenol blue)
6. Loading Dye
162
Connect the lids and set at 30 mins, 50 volts
o 1 hr: DNA fragments migrate further
8. Run gel at constant voltage until band separation occurs
163
Cap the gel tray to prevent electrocution
-Loaded with loading dye
8. Run gel at constant voltage until band separation occurs
164
Clue to prevent overpassing of agarose gel:
migration is 3.5 – 4.0 cm (close to 60% of the agarose gel or 75%)
