Using Gel Electrophoresis to Study Molecules

What is Gel Electrophoresis? A process that uses electricity to separate charged molecules: DNA fragments (double stranded) RNA (single stranded) Proteins negatively charged neutral (SDS added to give negative charge)

Components of Gel Electrophoresis: Gel Matrix (agarose or polyacrylamide) Electrophoresis Running Buffer (TAE or TBE) Sample Loading Buffer Gel Casting Tray with Comb Gel Box Power Supply Sample (DNA, RNA, or protein) Molecular Weight Marker/Ladder

Gel Matrix Fibrous matrix that acts as a molecular strainer to aid in the separation of molecules based on: Size larger molecules separate from smaller molecules Shape linear molecules separate from globular molecules Charge positively charged molecules separate from negatively charged molecules

Gel Matrix Material 1) Agarose Used in horizontal gel boxes to separate medium to large pieces of DNA and RNA Carbohydrate derived from seaweed; non-toxic Dissolved in a buffer solution (boiling required) Usually TAE (Tris, Acetic Acid, EDTA) or TBE (Tris, Boric Acid, EDTA)

Gel Matrix Material 2) Polyacrylamide Used in vertical gel boxes to separate smaller molecules such as proteins (mainly) Rarely used to separate very small pieces of DNA or RNA Cross-linked polymer of acrylamide; neurotoxin Uses a Tris/Glycine/SDS buffer system

Agarose Gel Concentrations Agarose can be used to separate … DNA pieces between 500 and 25,000 base pairs (bp). RNA pieces less than 5000 bases. Higher concentrations of agarose result in a tighter mesh. 0.6 – 0.7%: used for very large DNA molecules 0.8 – 1.0%: most common; used for restriction digests and plasmids 2.0 – 3.0%: used for much smaller DNA fragments Harder to prepare Sometimes polyacrylamide is a better choice

Sample Movement based on SIZE Small fragments move faster and farther from the well compared with larger fragments Single stranded RNA moves faster through the gel than double stranded DNA Fragments of similar size, charge, and shape will move at the same rate Short molecules move through matrix easily; travels further Large molecules require more effort to move through matrix; does not travel as far

Sample Movement based on CHARGE A sample of charged molecules is loaded into the sample wells and power is added to establish an electrical field. If the molecules have a net negative charge, they will move toward the anode (+ electrode: red). If the molecules have a net positive charge, they will move toward the cathode (- electrode: black).

Sample Movement based on CHARGE DNA and RNA are negatively charged due to their phosphate groups Which direction will the DNA or RNA move? They move toward the positively charged anode! RUNS TO RED!

Running Buffer Salt/Buffer solution: Salt supplies the ions needed to establish an electric field. Buffer stabilizes the pH to maintain the shape of the molecule being analyzed. TAE (Tris, Acetic Acid, EDTA; pH ~ 8.2) -- Used more often – cheaper TBE (Tris, Boric Acid, EDTA; pH ~ 8.2) Gels are usually run at 100-150 V (~35 mAmps)

Sample Loading Buffer Contains: Glycerol: Viscose chemical that adds density to sample so it sinks in well preventing the sample from floating out. Tracking Dyes: Adds color to sample so it can be seen to be loaded and tracked. Xylene Cyanol -- mid size dye Bromophenol Blue -- small size tracking dye (runs in front of DNA molecules) Orange G -- very small dye; usually used with PCR NOTE: these are NOT DNA stains

Gel Stains DNA and RNA molecules are colorless therefore they need to be stained in order to be seen Common Stains: Methylene Blue Ethidium Bromide (EtBr) SybrSafe Green - NEW

Gel Stains Methylene Blue: Stains DNA dark blue; visible under white light Very low sensitivity; requires higher quantities of DNA to be visible No health risks involved with its use; use in schools but not common in industry

Gel Stains Ethidium Bromide (EtBr): most commonly used stain binds to DNA and fluoresces orange under UV light extremely sensitive TOXIC!!! carcinogenic and mutagenic with chronic exposure! can be added to gel prior to casting or gel can be stained after the run.

binds to DNA and fluoresces green under UV light extremely sensitive Gel Stains SybrSafe Green: New stain binds to DNA and fluoresces green under UV light extremely sensitive SAFE --NOT TOXIC SybrSafe Green vs Ethidium Bromide

Molecular Weight Marker/Ladder A set of standards that are used to identify the approximate size of a molecules run on a gel during electrophoresis contain fragments of known sizes commercially purchased

Molecular Weight Marker/Ladder Can you estimate the size of the DNA bands?