AGAROSE GEL ELECTROPHORESIS Flashcards
Movement of dispersed, charged particles relative to a fluid under the influence of a spatially uniform electric field
ELECTROPHORESIS
Used in the separation of DNA, RNA, or proteins based on – and –
the molecular size and net electrical charge
attraction or repulsion between charged particles
Electrostatic 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
Electrostatic Force (Fe) equation
𝐹𝑒 = 𝐸 × 𝑞
E = electric field (V/m)
q = charge of particles (C)
Drag Force (Fd) equation
𝐹𝑑 = 𝑓 × 𝑣
f = frictional coefficient
v = velocity of the particle (m/s)
Electrophoretic
Mobility (μ)
𝜇 = 𝑞/𝑓 = 𝑣/𝐸
Velocity
of the
particle
𝑣 = (𝑞 × 𝐸)/𝑓
Charges will migrate
to their opposite
pole
Net Charge
Smaller molecules
will travel farther
Molecule
size
Denatured DNA will
migrate more
predictably according
to size
Molecule
Shape
𝑉 = 𝐼𝑅
Applied
Voltage
composition and
properties of the
buffer affects
electrophoretic
mobility
The buffer
Pore size of the gel
will determine the
ease of movement of
differently sized
macromolecules
The Gel
Sulfonated polysaccharide polymer
from seaweed
Agarose Gel
Agarose Gel
- Linear polymer of ________
consisting: - ___________
- ___________
agarobiose
- 1,3-linked-β-D-galactopyranose
- 1,4-linked-3,6-anhydro-α-Lgalactopyranose
Agarose Gel
- Process:
- Dried powder is dissolved in hot running buffer
- Cooled between ____ then poured to a casting tray
- ____ is inserted to create wells for the sample
- Solidifies at ____
- Concentration dictates the size of the spaces in the gel (pore size)
- Higher concentration = _____
- 55-65 oC
- Comb
- ~40 oC
- smaller pores
- Better for proteins
- Components are synthetic, causing less differences in batches from different sources
*Powdered form is a potent neurotoxin
*Difficult gel preparation (thinner gels)
Polyacrylamide
- 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 (______ for nucleic acids)
what am I and what ph for nucleic acids?
- RUNNING Buffer
- pH 8.0
Henderson Hasselbach Equation
𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔 ([𝐴−]/[𝐻𝐴])
T or F:
Increase in concentration of the buffer causes increased conductivity and offers greater pH stability
T
T or F:
Greater conductivity results in lesser heat generation at a given voltage
F - greater
Solution: Higher buffer concentrations ran at a higher voltage
F - lower
- Most commonly used for DNA electrophoresis
- DNA migrates faster in TAE
- More stable when stored
TRIS Acetate EDTA
(TAE)
- Greater buffering capacity
- Stock solutions are prone to
precipitation
Tris borate EDTA
(TBE)
Tris borate EDTA (TBE)
* Causes a gradient
concentration difference
that distorts migration
patterns (_________)
salt wave
Buffer additives
*________agents are commonly used for nucleic acids to break down H-bonds between complementary strands or within the same strand of DNA or RNA
*____
*____
- Denaturation
- Formamide
- Urea
- Contain a colored dye and a density agent
Loading Buffer
_____ to increase the density of the
sample in comparison to the buffer
Density agents (e.g. Ficoll, sucrose, or
glycerol)
_____ monitors the progress of
migration (e.g. Bromophenol blue)
Tracking dye
- Agarose gels are ran horizontally in
acrylic containers (_____) - ____ in the gel compartment are connected to a power supply
- The gel is placed in the middle and
submerged in the running buffer, filling
both compartments
- gel boxes
- Electrodes
Gel Loading- Loading the samples
✓ Dilute the samples in your ____ 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
- loading buffer
- 25 μL
After electrophoresis, the bands are visualized using dyes that specifically associate with nucleic acids (fluorescent dyes and silver stain)
Dyeing your gel
- Intercalating agent
- Used to be the most widely used dye
- Highly carcinogenic
Ethidium bromide
Ethidium bromide
Emits visible light at ____
(orange) when excited at UV
light of ____
- 590 nm
- 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
SYBR Green
double-stranded DNA (used in
RT-PCR)
SyBr Green I
SYBR Green
Single-stranded DNA or RNA
staining
SyBr Green II
SYBR Green
Can be used for both DNA and
RNA staining
SyBr Gold
SYBR Green
Variant of SyBr Green that has
been deemed as nonhazardous waste
SyBr Safe
- Intercalating agent
- Cannot penetrate the cell membrane
- Higher sensitivity than EtBr
- Can be used for dsDNA,
ssDNA, or RNA
Gel Red
- Similar absorption and
emission spectra as EtBr - Can be added to the gel mix
like SyBr Green - Cheaper than SyBr Green
Gel Red (gihapon)
- 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
