Module 2.2- Laboratory Techniques (Gel Electrophoresis)

Gel electrophoresis

• A laboratory technique that separates charged molecules like DNA, RNA, and proteins based on their size, charge, and conformation

  • verifies that we have amplified our DNA properly

Uses of electrophoresis

• Analysis of DNA and proteins

  • Purity, integrity, fragmentation, synthesis efficiency

  • Quantitation

• Separation and purification of DNA and proteins

• Detection of sequences of interested (e.g. blotting)

Common gels

Agarose and polyacrylamide

Gel electrophoresis consists of…

1. Electric field power supply

2. Porous matrix gel

3. Conductive medium buffer solution

How gel electrophoresis works

• DNA & RNA are negatively charged → travel to the positive end

• The molecules travel through the gel (porous matrix) at a rate inversely proportional to their size

• Small DNA will travel faster (and further) than a larger DNA molecule

Agarose 

• A type of gel used for gel electrophoresis 

  • A complex sugar derived from seaweed

  • In electrophoresis, these gels are run horizontally

  • The higher the ________ concentration, the smaller the pore sizes are and the DNA migrates a smaller distance (typically used at concentrations of 0.5 – 2%); pores are not uniform in size → 50-200 nm

  • This can be used for DNA fragments of 50 – 20,000 bp in size (good for large)

  • Non toxic

Polyacrylamide 

• A type of gel used for gel electrophoresis 

  • Acrylamide is made by digesting acrylonitril with nitrile hydratase and polyacrylamide is made by the chemical crosslinking of acrylamide and bis-acrylamide

  • The pore sizes are determined by the ratio of acrylamide to bis-acrylamide; pores are uniform in size

  • The gels are usually made at a concentration of 6 – 15%

  • These gels have a very high resolving power for small 5 – 500 bp fragments of DNA

  • Good for analyzing single-stranded DNA (and proteins)

  • Potent neuro-toxicity

Porosity

A measure of how many pores there are in a given agarose gel – with a greater gel concentration, porosity decreases

Effect of smaller pore sizes in gel composition

Make it harder for DNA to migrate through the gel during electrophoresis

Pore size distribution

• The set of the sizes of pores that exist in the gel

  • In a more concentrated agarose gel, the largest pore that exists is likely to be smaller than in a less concentrated gel

  • More pores and larger pores will allow for larger DNA molecules to effectively migrate through the gel

  • Less and smaller pores will allow for greater distinction between smaller DNA molecules

DNA ladder/marker

A solution of double-stranded DNA fragments whose molecule weights (and number of base pairs) are known and standardized→ usually cut plasmid or bacteriophage DNA

Molecular size of DNA

Rate of migration is inversely proportional to log10 of the number of base pairs

Conformation of DNA

Supercoiled vs linear (good controls to include)

Presence of EtBr in gel or buffer

Intercalation → decrease in negative charge + increase in stiffness & length → rate retarded by ~15%

Applied voltage

For max resolution of > 2 kb fragments → 1-5 V/cm

Electrophoresis buffer

Composition and ionic strength of the buffer (must use same buffer for gel) → no ions→ no DNA movement, too much ions → too much heat !!

• TAE: Tris-Acetate EDTA: faster migration, higher resolving power for larger DNA, lower buffering capacity

• TBE: Tris-Borate EDTA

Tracking dyes

• Dyes move towards anode at a predictable rate

  • bromophenol blue: same speed as DNA fragments consisting of 300 bp

  • xylene cyanol: same speed as DNA fragments consisting of 4000 bp

• Rate independent of agarose concentration 0.5-1.4%

• Tracking dyes do not directly influence the visibility of DNA fragments, comparing the position of a DNA fragment in the gel to the position of these dyes allow technicians to “see” the approximate number of nucleotides the DNA fragment contains

How to visualize DNA

• Gel is stained with a fluorescent dye that binds to the DNA

• Placed on an ultraviolet transilluminator which will show up the stained DNA as bright bands

Ethidium bromide (EtBr)

• Flat molecule that fits between adjacent base pairs (intercalates) in the DNA double helix→ 1–5 ng (Ex ~260 nm/Em 590 nm)

• Its fluorescence is significantly higher when intercalated (inserted between layers) than it is in aqueous solution

UV transilluminator

A device used in gel electrophoresis to visualize DNA, RNA, or proteins that have been separated by size