MIDTERM LAB: ELECTROPHORESIS Flashcards
Most practical and recently use of resolution & detection of nucleic acids
- Separation of molecules in electric current.
ELECTROPHORESIS
ability to distinguish separate individuals components of nucleic acids.
RESOLUTION
ELECTROPHORESIS Purpose: Separate molecules by ?
SIZE AND CHARGE
The negative pole.
CATHODE
POSITIVE POLE
ANODE
NEGATIVELY CHARGED MOLECULE
ANION
POSITIVELY CHARGED MOLECULE
CATION
anions travel to the ?
anode
cations travel to the ?
cathode
Nucleic Acids (NA) moves from
NEGATIVE TO POSITIVE POLE
When DNA is applied to a macromolecular case or gel, its
migration under the pull of current is
IMPEDED
- May move rapidly in large pore size
(Low gel concentration) - May move slowly in small pore size
(High gel concentration)
LARGE NUCLEIC ACID FRAGMENTS
- May move rapidly in large pore size
and small pore size.
SMALL NUCLEIC ACID FRAGMENTS
- to separate SMALL NUCLEIC ACID FRAGMENTS, it should be in______________(pores become small)
HIGH GEL CONCETRATIONS
The CONCENTRATION OF GEL/BUFFERS will affect the _____ of fragments of different size ranges.
RESOLUTION
sample are spotted in the center of the paper, high voltage is applied and the spots migraine according to their
charges
PAPER
separates AMPHOTERIC molecules (have both acidic & basic properties/alkaline properties (protein)) according to their charged as defined by the pKa values of proton-accepting sites within a molecule
TUBE (ISOELECTRIC FOCUSING)
- most frequently used
- horizontal/vertical
- sample is introduced into the gel at
the cathode end and migrates with the current toward the anode
SLAB GELS
separation technique of molecules and ions performed in a narrow capillary tube structure using electric current
CAPILLARIES
Provide resistance to the movement of molecules under the force of electric current forming bands.(DNA fragments)
GEL SYSTEMS
- The movement is impeded in the gel and will form it.
o Dependent to the speed of migration.
BANDS
Serve as support medium for analysis of the separated components
GEL
As the gel concentration increases, the movement of particles (DNA fragments) ______
SLOWS
CRITERIA FOR A GOOD GEL:
- Unaffected by electrophoresis
- Simple to prepare
- Amenable to modification
TYPES OF GEL:
- AGAROSE
- POLYACRYLAMIDE
- COMPOSITE AGAROSE-POLYACRYLAMIDE
- Polysaccharide polymer of AGAROBIOSE
- Is composed of D - galactose
- Extracted from SEAWEEDS
- Component in agar
- Usage: Powder is suspended in buffer, heated and poured into mold.
- Cooled 55 – 56 °C
- Formulations:
o Small DNA (50-500 BP) – 2-3%
o Large DNA(2000 – 5000 BP) - Easier casting
- Gel is NEGATIVELY CHARGED – thus surrounded by buffers
- Usually on HORIZONTAL SETUPS
AGAROSE GEL
- SYNTHETIC
- Repeating units: Acrylamide + methylene bisacrylamide
- Higher resolution than agarose
- Mobility can depend on DNA sequence (agarose do not, solely on size only.
- For very small DNA,SSDNA, RNA, proteins
- Forms: Powdered form (neurotoxic), commercial solutions, and performed gels.
- Usually in VERTICAL setups
- Requires at polymerization catalysts (for gel to solidify)
POLYACRYLAMIDE GEL
POLYACRYLAMIDE GEL Requires at polymerization catalysts (for gel to solidify) such as?
- AMMONIUM PERSULFATE
- TETRAMETHYLENEDIAMINE
- LIGHT ACTIVATION
- Carry the current and protect samples during electrophoresis.
- Composed of weak acid+ its conjugate base
- Uncontrolled ph may damage the sample
- High concentration means higher conductivity, thus we need to lower the voltage.
BUFFERS
BUFFERS FOR DNA?
- TRIS BORATED EDTA (TBE)
- TRIS ACTETATE EDTA (TAE)
- TRIS PHOSPHATE EDTA (TPE)
BUFFERS FOR RNA
- 10nM SODIUM PHOSPHATE
- MOPS BUFFER
BUFFER ADDITIVES THAT BLOCK HYDROGEN BONDING SITES
FORMAMIDE + HEAT
BUFFER ADDITIVES THAT PREVENT FOLDING
UREA + HEAT
__________prevents hydrogen bonds and formation of HETERODUPLEXES – FOLDED SSDNA/RNA forming HAIRPIN-LIKE structures that will impair MIGRATION.
DENATURING AGENTS
Gel is positioned horizontally
Gel is submerged throughout the loading and electrophoresis process
Samples are located into wells at one end of the gel
HORIZONTAL/SUBMARINE GEL (AGAROSE GEL are commonly used)
Gel is positioned vertically
Samples are loaded into wells at the TOP OF THE GEL
Spacers: determine the thickness of the gel
VERTICAL GEL (POLYACRYLAMIDE GEL
are often used)
used to create wells in the gel where sample can be loaded
ELECTROPHORESIS COMBS
determine the CAPACITY of the well for the sample
SIZE OF TEETH
determine the number of wells
NUMBER OF TEETH
TYPE OF ELECTROPHORESIS COMB:
-WELLS SEPARATED BY AN “EAR” OF THE GEL.
- placed on TOP of the gel.
- evenly spaced teeth = creates well of uniform size & is suitable for general purposes; DNA/protein electrophoresis
REGULAR COMBS
TYPE OF ELECTROPHORESIS COMB:
-WELLS IMMEDIATELY ADJACENT
-Placed upside-down, after polymerization Is placed side-down at top.
- teeth resembling a pointed/shark’s tooth
- for preventing sample leakage/ improving loading accuracy
HOUNDSTOOTH/SHARKSTOOTH
RUNNING A GEL
➢ Use the proper gel concentration
for sample size range
➢ Use the proper comb (well) and
size
➢ Load sample mixed with: TRACKING DYE & DENSITY AGENT
to monitor the progress of the electrophoresis run.(Migrate at specific speeds in a given gel concentration.)
EX: bromophenol blue; xylene cyanol green
TRACKING DYE (dye + density agent)
allows nucleic acids to fall in the sample wall. (Allow sinking of the nucleic acid to the wall instead of diffusing to the buffer.)
EX: sucrose, ficoll, glycerol
DENSITY AGENT
Action of putting the isolated nucleic acid in wells.
GEL LOADING
Prior to loading, THINGS SHOULD
BE ADDED:
- Tracking dyes
- Density agent
ELECTROPHORESIS WILL TERMINATE IF:
DYE APPROACHES TO THE END OF
THE GEL, OR
OBTAINED THE DESIRED DISTANCE
Visualization of bands after electrophoresis
DETECTION SYSTEMS
to make the separated molecules
visible
STAIN
For visualization of bands:
- UV TRANSILLUMINATOR
- FLUORESCENCE IMAGING SYSTEM
- GEL DOCUMENTATION SYSTEM
COMMONLY USED STAINS
- FLUORESCENT DYES
- SILVER STAINS
FLUORESCENT DYES:
stack (intercalate) between adjacent nitrogen based in double stranded nucleic acids.
INTERCALATING AGENTS
FLUORESCENT DYES:
sits in the minor groove of the double helix
Minor groove – binding dyes
Preferred for real time PCR
SYBR GREEN
- originally developed for protein
visualization
Fixed with methanol + acetic acid
insoluble in black silver after formaldehyde
SILVER STAINS
SILVER STAINS 2 METHODS
- SILVER DIAMINE/AMMONIACAL SILVER
-SILVER NITRATE
- For very large DNA fragments (50k
to 250 + kBP) - Pulses of current applied to the gel
in alternating dimensions enhance
migration.
PULSE-FIELD GEL ELECTROPHORESIS
PULSE-FIELD GEL ELECTROPHORESIS TYPES:
- Alternating positive and negative poles, provides good resolution (>800kb)
FIELD INVERSION GEL ELECTROPHORESIS (FIGE)
PULSE-FIELD GEL ELECTROPHORESIS TYPES:
- Transverse angle reorientation of
poles in a vertical gel.
TRANSVERSE ALTERNATIVE GEL
ELECTROPHORESIS (TAFE)
PULSE-FIELD GEL ELECTROPHORESIS TYPES:
- Alternating polarity in an electrode
array; DNA molecules as large as 2Mb can be well separated.
CONTOUR- CLAMPLED HOMOGENOUS
ELECTRICAL FIELD (CHEF)
PULSE-FIELD GEL ELECTROPHORESIS TYPES:
- Rotating gel with fixed poles; 50kb to
6000 KB
ROTATING GEL ELECTROPHORESIS
(RGE)
PULSE FIELD GEL ELECTROPHORESIS APPLICATIONS
Molecular typing and identification of pathogens
Gold standard method of some bacterial ids
Genotyping for antibiotics resistance of:
o Staphylococcus
aureus
o Pseudomonas
aeruginosa
o Acinetobacter
baumannii
o Mycobacterium
tuberculosis
Epidemiological studies
Relationship of strains of same
DNA fingerprinting of viruses
Separates solutes by charge/mass ratio
Separated nucleic acids,
metals, anions, carbohydrates, pharmaceuticals
Thin glass (fused silica)
capillary 30-100cm X 25-100um
internal diameter
Linear or cross-linked polyacrylamide or other linear polymers used for sieving
Separation based on size
CAPILLARY ELECTROPHORESIS
