AGE Flashcards
Electrophoresis
Movement of dispersed, charged particles relative to a fluid under the influence of a spatially uniform____
electric field
Used in the separation of (3) based on the _____and _____
DNA, RNA, or proteins
molecular size and net electrical charge
- electric field,
- migration
Most biomolecules exist as electrically-charged particles
ELECTRO
PHORESIS
- attraction or repulsion between charged particles
Electrostatic Force
Electrostatic force =
Coulomb force
- Resistance to motion when the surface of one object comes in contact with another.
Friction Force
- Force exerted by the medium on particles in a direction opposite to particle movement
Electrophoretic Retardation Force
is a force that slows down the movement of charged particles in a solution.
It occurs when charged particles move in one direction, while the ions around them move in opposite direction
Electrophoretic retardation force
- solute response to applied electric field
Electrophoretic mobility
_____acting on an object moving in a fluid oppose the motion. Ex.
Aerodynamic force that opposes an aircraft’s motion through the air.
Drag forces
ELECTROSTATIC FORCE (Fe)
DRAG FORCE (Fd)
E = electric field (V/m)
q = charge of particles (C)
f = frictional coefficient
v = velocity of the particle (m/s)
NET CHARGE
Charges will migrate to their…
opposite pole
MOLECULAR SIZE
Smaller molecules will…
travel farther
MOLECULE SHAPE
Denatured DNA will migrate…
more predictably according to size
APPLIED VOLTAGE
Formula
V= IR
Composition and properties of the ___ affects electrophoretic mobility
THE BUFFER
THE GEL
______ of the gel will determine the ease of movement of differently sized macromolecules
Pore size
FACTORS AFFECTING A.G.E.
NET CHARGE
MOLECULAR SIZE MOLECULE SHAPE
APPLIED VOLTAGE
THE BUFFER
THE GEL
AGAROSE GEL
Sulfonated polysaccharide polymer from…
seaweed
AGAROSE GEL
Linear polymer of_____ consisting:
(2)
It is a disaccharide (combination of two monosaccharides).
agarobiose
1,3-linked-ß-D-galactopyranose
1,4-linked-3,6-anhydro-a-L-galactopyranose
______ is made up of a repeating unit of D-galactose and 3, 6-anhydro-L-galactopyranose joined by 1-4 linkages.
Agarobiose
Agarobiose is made up of a repeating unit of______ and _____ joined by_____
D-galactose and 3, 6-anhydro-L-galactopyranose
1-4 linkages.
Agarose
Process:
Dried powder is dissolved in hot running____
Cooled between____ then poured to a casting tray
_____is inserted to create wells for the sample
_____(comb appearance)
Solidifies at____
buffer
55-650C
Comb
Comb-molder
~40C
_______ dictates the size of the spaces in the gel (pore size)
Higher concentration =_____
Concentration
smaller pores
Separation of linear, double-stranded DNA molecules in TAE agarose with regular voltage below___
5 V/cm.
- Better for proteins
- Components are synthetic, causing less differences in batches from different sources
- Higher resolution of smaller fragments
Polyacrylamide
- Powdered form is a potent neurotoxin
- Difficult gel preparation (thinner gels)
Polyacrylamide
- organic chemicals: pharmaceuticals and carbohydrates,
inorganic anions and metals
Capillary
- Increased sensitivity
- Immediate detection - multiple color detection systems
- Decreased labor and run time
Capillary
Instruments used are very costly compared to AGE
Capillary
Carries the current and protects the samples
Weak acid and its conjugate base able to take up or release protons to maintain a constant pH (pH 8.0 for nucleic acids)
Running buffer
In order to change the pH of a buffered solution by 1 point, the acidic or basic form of the buffer
must be brought to a concentration 1/10 that of the other form - basically like diluting to 1/10 to change the pH
Henderson-Hasselbach Equation
^ Buffer concentration =_____
Increase in concentration of the buffer causes increased_____ and offers greater____ stability
Greater conductivity results in greater heat generation at a given voltage
Solution:…
^ HEAT
conductivity; pH
Higher buffer concentrations ran at a lower voltage
Most commonly used for DNA electrophoresis
DNA migrates faster
*More stable when stored
TRIS Acetate EDTA (TAE)
Greater buffering capacity
Stock solutions are prone to
precipitation
Causes a gradient concentration difference that distorts migration patterns (salt wave)
Tris borate EDTA (TBE)
-Denaturation agents are commonly used for nucleic acids to break down H-bonds between complementary strands or within the same strand of DNA or RNA
BUFFER ADDITIVES
BUFFER ADDITIVES
• Formamide
• Urea
• Results in long, straight, unpaired chains that have a more predictable migration through the gel
Buffer additives
(e.g. Ficoll, sucrose, or glycerol) to increase the density of the sample in comparison to the buffer
monitors the progress of migration (e.g. Bromophenol blue)
- Density agents
-Tracking dye
(e.g. Ficoll, sucrose, or glycerol) to increase the density of the sample in comparison to the buffer
monitors the progress of migration (e.g. Bromophenol blue)
- Density agents
-Tracking dye
EQUIPMENT
______are ran horizontally in acrylic containers (gel boxes)
______in the gel compartment are connected to a power supply
The gel is placed in the middle and submerged in the_____, filing both compartments
Agarose gels
Electrodes
running buffer
GEL LOADING
LOADING THE SAMPLES
✔ Dilute the samples in your loading buffer prior to loading
✔ Know the capacity of the well (usually,_____ is a safe volume for a >5 well format)
✔ Depress the plunger of the micropipette before loading and do not let go until you are done and out of the buffer
✔ Make sure your tip is within the well before loading
✔ When withdrawing the tip, try to go straight up
✔ Take care to not pierce the gel all the way down
25 μL
DETECTION SYSTEMS
DYEING YOUR GEL
After electrophoresis, the bands are visualized using dyes that specifically associate with nucleic acids (2)
fluorescent dyes and silver stain
- Intercalating agent
- Used to be the most widely used dye
- Highly carcinogenic
ETHIDIUM BROMIDE
Ethidium Bromide
- Emits visible light at ______when excited at UV light of____
590 nm (orange)
300 nm
- Sits on the minor groove of the double helix
- 25-100x more sensitive than EtBr
- Can be added to the gel mix
- Requires ****special optical filters
SYBR GREEN
• Variant of SyBr Green that has been deemed as nonhazardous waste
- SyBr Safe
• Can be used for both DNA and RNA staining
- SyBr Gold
• Single-stranded DNA or RNA
staining
- SyBr Green Il
• double-stranded DNA (used in rt-PCR)
- SyBr Green I
- Intercalating agent
- Cannot penetrate the cell membrane
- Higher sensitivity than EtBr
GEL RED
- Can be used for dsDNA, ssDNA, or RNA
- Similar absorption and emission
spectra as EtBr - Can be added to the gel mix like SyBr Green
Cheaper than SyBr Green
• 136 USD = 7.6k (for 0.5 mL before taxes and shipping)
• SyBr Green 155 USD = 8.7k (for 0.5 mL before taxes and shipping)
Gel Red
- Not an intercalating agent and is thus not toxic to humans
- Considered a biohazard
- More complicated than fluorescent stains
- Increased sensitivity
- Most useful in protein analysis
SILVER STAIN
_______is usually 10-100 times more sensitive than Coomassie Blue staining, but it is more complicated.
Faint but still visible bands on this gel contain less than 0.5 ng of protein!
Silver staining
