Transcript Gel Electrophoresis

What is Gel Electrophoresis?
 A procedure that separates molecules based on size and
charge.
 Uses an electric field
 Molecules move through gel made of agar or
polyacrylamide
Simple Procedure
 Molecules are dispensed into a well in the gel material.
 Gel is placed in an electrophoresis apparatus
 Electric current is applied to the contents of the
apparatus
 In the gel:
 Larger molecules move more slowly
 Smaller molecules move faster
 The different sized molecules form distinct bands on
the gel.
Types of Gel
 Agarose – DNA and protein
 Polyacrylamide – Protein and DNA
 Starch - protein
Agarose Gel
 Easy to cast, handle, store or dispose
 Used to separate DNA fragments ranging from 50 base
pair to several megabases (millions of bases).
 Distance between DNA bands is determined by the
percent agarose in the gel.
 0.7% (large 5–10kb DNA fragments) and 2% (small
0.2–1kb fragments) agarose.
 Low percentage gels - very weak and may break
 High percentage gels - brittle and don’t set evenly
Polyacrylamide
 used for separating proteins ranging in size from 5 to
2,000 kDa - Uniform pore size
 Pore size is controlled by controlling the concentrations
of acrylamide and bis-acrylamide powder used in
creating a gel.
 used to separate different proteins or isoforms of the
same protein into separate bands and small DNA
 gels are made in 6%, 8%, 10%, 12% or 15%.
 percentage chosen depends on the size of the protein
 smaller weight = higher gel
Starch
 Partially hydrolysed potato starch – protein
electrophoresis
 Non-denatured proteins are separated according to
charge and size.
 They are visualised using Napthal Black or Amido
Black staining.
 Typical starch gel concentrations are 5% to 10%
Nucleic Acid Electrophoresis
 analytical technique used to separate DNA or RNA fragments by
size.
 an electric field induces the nucleic acids to migrate toward the
anode
 smaller fragments end up nearer to the anode
 DNA is frequently cut into smaller fragments using a DNA
restriction endonuclease (or restriction enzyme) or even PCR
 Fragment size determination is typically done by comparison to
commercially available DNA markers containing linear DNA
fragments of known length
 agarose (for relatively long DNA molecules) and polyacrylamide
(for high resolution of short DNA molecules
Protein Electrophoresis
 proteins, unlike nucleic acids, can have varying charges and
complex shapes
 proteins are usually denatured in the presence of a detergent such
as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP)
 the rate at which the resulting SDS coated proteins migrate in the
gel is relative only to its size and not its charge or shape.
Protein Electrophoresis cont.
 Proteins are usually analyzed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDSPAGE), by native gel electrophoresis, by quantitative
preparative native continuous polyacrylamide gel
electrophoresis (QPNC-PAGE), or by 2-D
electrophoresis.
Gel Condition
 Denature - the native structure of
macromolecules that are run within the gel
is not maintained.
 denatures the native structure of a protein
 Native - separation method typically used
in proteomics and metallomics.
 does not use a charged denaturing agent.
Buffers
 nucleic acids - Tris/Acetate/EDTA (TAE),
Tris/Borate/EDTA (TBE).
 TAE - lowest buffering capacity but best resolution for
larger DNA. This means a lower voltage and more
time, but a better product.
 Xylene cyanol and Bromophenol blue are common
dyes found in loading buffers
Visualization
 DNA may be visualized using ethidium bromide
 SYBR Green I – more expensive, quicker, safer
Ethidium Bromide
 light sensitive and is stored in a brown bottle covered in
tin foil (aluminum foil).
 fluoresces under UV light when intercalated into the
major groove of DNA (or RNA)
 any band containing more than ~20 ng DNA becomes
distinctly visible
 protein may be visualised using silver stain or Coomassie
Brilliant Blue dye.
 Mutagen
 ALWAYS wear gloves
Areas of Application
 used in forensics, molecular biology, genetics,
microbiology and biochemistry.
 results can be analyzed quantitatively by visualizing the
gel with UV light and a gel imaging device
 the intensity of the band or spot of interest is measured
and compared against standard or markers loaded on
the same gel.
 the measurement and analysis are mostly done with
specialized software.
Lab Procedure
 Make Ethidium Bromide
 Make buffer
 Add agarose to buffer and microwave
 Pour gel after setting comb
 Pour remaining buffer + EtBr in apparatus
 Load gel (Black to Red 60 – 100 V)
 Run gel
 Kodak Moment 
 Discard waste product safely
Making EtBr
 1 g of Ethidium Bromide in 100 ml of dH20 in DARK
colored bottle of transparent glass bottle wrapped in
aluminum foil.
 Dissolve completely (use Stir bar)
 Use caution
 Wear gloves at all times
 Discard all EtBr related waste to biohazard
Making Buffer
 Calculation: Total of 300 ml of solution
 15 ml of TBE + 285 ml dH2O
Making Gel
 Calculations: 0.8% agarose gel in 100 ml of solution
 Mix 0.8 g of agarose gel in 100 ml of buffer solution.
 Microwave until agarose dissolves completely in
buffer.
 Let it cool (not solidify).
 Pour!
Preparing the DNA sample
 10 microliter DNA + 2 microliter tracking dye in
microcentrifuge tube.
 Ladder: 1 microliter concentrated ladder per 9
microliter sterile water
 Load all contents in separate wells in gel.
Setting up the apparatus
 5 microliter Ethidium Bromide + 100 microliter TBE
solution in electrophoresis apparatus
 Plug apparatus into the electric outlet
 Put gel in the apparatus (wells being in the black side)
 DNA will travel from Black to Red
 Set V to 60 or 100
 Run DNA for about 30-40 minutes
What is the safe way to
discard the waste?
 Put all EtBr related waste in the biohazard bin.
 Discard buffer from the apparatus into biohazard bottle.
 Wash gel in sink before throwing in the trash