Gel Electrophoresis & Gel Loading Practice

Gel Electrophoresis The process by which electricity is used to separate charged molecules (DNA fragments, RNA, and proteins) based on there size, shape, and charge.

What you should already know… MoleculeChargeBehavior DNANegativeMoves to positive RNANegativeMoves to positive ProteinsPositive Negative Neutral Moves to negative Moves to positive No movement CarbohydratesNeutralNo movement LipidsNeutralNo movement Remember that opposite charges attract so if a molecule is negatively charged it will move towards the area with a positive charge DNA (negative charge) runs to red (red=positive charge) Wells

How it Works  Molecules are separated by an electrical current moving the molecules through an agarose gel  Electrical Current: Establishes electric field between the positive and negative electrodes  Causes molecules to move from well (where samples are loaded) through the gel  Positive molecules move toward negative end  Negative molecules move toward positive end  Agarose Gel: Acts as a “Molecular Strainer”  Creates a gel matrix  Smaller molecules pass more easily through the tiny spaces in the gel matrix and therefore run faster and farther than larger molecules Well

HINT!!!  Picture all of your friends running through a jungle. Your tall friends will probably get caught more easily on low hanging vines or branches and may struggle to get through particularly dense areas. Your short friends, however, will easily elude low hanging vines or branches and will be able to get through the areas with the dense vegetation that your taller friends struggled with. Your smaller friends (smaller molecules) will be able to travel farther and faster than your taller friends (larger molecules) because they won’t be caught by the dense vines and trees (gel matrix) that your tall friends will be slowed down by.

How We See the Results Methylene blue – a staining dye/indicator that interacts with nucleic acid molecules and proteins, turning them to a very dark blue color Used to see where samples are when loading them in a gel Ethidium bromide – a DNA stain (indicator); glows orange when it is mixed with DNA and exposed to UV light; abbreviated EtBr Used to see how molecules were separated after running gel Methylene Blue Ethidium Bromide

Contents of Gel DNA Samples Ladder, or sample containing DNA fragments of known length/size (in base pairs) Used to estimate size of/base pair length of isolated DNA fragments or other DNA samples run on the same gel

Concept Check!  Where, on this gel are the largest molecules (in this case DNA fragments)? The smallest? Largest Smallest

Gel Loading

Loading Samples: Good

Loading Samples: BAD Micropipette tip punched through the gel