Horizontal Horizontal Gel Electrophoresis for Gel Electrophoresis for

Nucleic AcidsNucleic Acids

Introduction for Introduction for

Restriction Enzyme Digest and Restriction Enzyme Digest and Polymerase Chain Reaction LabsPolymerase Chain Reaction Labs

How do we visualize DNA?How do we visualize DNA? Agarose Gel ElectrophoresisAgarose Gel Electrophoresis

Complete a Gel Electrophoresis simulation at:

http://gslc.genetics.utah.edu/units/biotech/gel/

Important Characteristics of DNA StructureImportant Characteristics of DNA Structure

Nucleus is the Nucleus is the “command “command center”center”

ChromosomesChromosomes Double-strandedDouble-stranded DNA helixDNA helix Encoded Encoded

information in the information in the form of bases = form of bases = “genetic code”“genetic code”

Important Characteristics of DNA StructureImportant Characteristics of DNA Structure

Genetic code:Genetic code: GuanineGuanine AdenineAdenine CytosineCytosine ThymineThymine

Phosphate-Sugar Phosphate-Sugar backbonebackbone NOTENOTE

Phosphate groups Phosphate groups are negatively are negatively chargedcharged

The mass:charge The mass:charge ratio is the same for ratio is the same for all sizes of DNA all sizes of DNA fragmentsfragments

Agarose Gel ElectrophoresisAgarose Gel Electrophoresis

Electrolysis:Electrolysis: the splitting of water using the splitting of water using electricityelectricity current splits water into hydrogen ions (Hcurrent splits water into hydrogen ions (H++) )

and hydroxyl ions (OHand hydroxyl ions (OH--))

ElectrophoresisElectrophoresis:: a method of separating a method of separating charged molecules in an electrical field; charged molecules in an electrical field; DNA has an overall negative chargeDNA has an overall negative charge

Used to separate DNA fragments by sizeUsed to separate DNA fragments by size

Components of an Electrophoresis SystemComponents of an Electrophoresis System

Power supply and chamberPower supply and chamber, a source of negatively , a source of negatively charged particles with a cathode and anodecharged particles with a cathode and anode

BufferBuffer,, a fluid mixture of water and ions a fluid mixture of water and ions

Agarose gelAgarose gel, a porous material that DNA migrates , a porous material that DNA migrates throughthrough

Gel casting materialsGel casting materials

DNA ladderDNA ladder, mixture of DNA fragments of known , mixture of DNA fragments of known lengthslengths

Loading dyeLoading dye, contains a dense material and allows , contains a dense material and allows visualization of DNA migrationvisualization of DNA migration

DNA StainDNA Stain, allows visualizations of DNA fragments , allows visualizations of DNA fragments after electrophoresisafter electrophoresis

Buffer

Dyes Power Supply

+

-

Agarosegel

Cathode

Anode

Bio-Rad’s Electrophoresis Equipment

Precast Ready Agarose Gel

Power Supplies

Electrophoresis BufferElectrophoresis Buffer

TAE (Tris-acetate-EDTA) and TBE (Tris-borate-TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are the most common buffers for duplex EDTA) are the most common buffers for duplex DNADNA

Establish pH and provide ions to support Establish pH and provide ions to support conductivityconductivity

Concentration affects DNA migrationConcentration affects DNA migration Use of water will produce no migratonUse of water will produce no migraton High buffer conc. could melt the agarose gelHigh buffer conc. could melt the agarose gel

New New Sodium Borate (SB)Sodium Borate (SB) buffer allows gels to buffer allows gels to be run at higher voltages in less time than be run at higher voltages in less time than traditional bufferstraditional buffers

Agarose GelAgarose Gel A porous material derived from red A porous material derived from red

seaweedseaweed

Acts as a sieve for separating DNA Acts as a sieve for separating DNA fragments; smaller fragments travel fragments; smaller fragments travel faster than large fragmentsfaster than large fragments

Plinko ModelPlinko Model

Concentration affects DNA migrationConcentration affects DNA migration

Low conc. = larger poresLow conc. = larger pores better better resolution of larger DNA resolution of larger DNA fragmentsfragments

High conc. = smaller poresHigh conc. = smaller pores better resolution of smaller DNA better resolution of smaller DNA fragmentsfragments

1% agarose

2% agarose

Loading DyeLoading Dye DNA samples are loaded DNA samples are loaded

into a gel AFTER the tank into a gel AFTER the tank has been filled with buffer, has been filled with buffer, covering the gelcovering the gel

Contains a dense Contains a dense substance, such as substance, such as glycerolglycerol, to allow the , to allow the sample to "fall" into the sample to "fall" into the sample wellssample wells

Contains one or two Contains one or two tracking dyestracking dyes, which , which migrate in the gel and migrate in the gel and allow monitoring of how far allow monitoring of how far the electrophoresis has the electrophoresis has proceeded. proceeded.

DNA StainingDNA Staining

Allows DNA Allows DNA visualization after visualization after gel electrophoresisgel electrophoresis

Ethidium BromideEthidium Bromide

Bio-Safe DNA Bio-Safe DNA stainsstains In gel stainingIn gel staining

Agarose Gel

DNA Fragments

Frequent MisconceptionsFrequent Misconceptions

Each band on the gel represents a single DNA Each band on the gel represents a single DNA strand...strand...NOT TRUE.NOT TRUE.

A single band/position in a lane contains A single band/position in a lane contains only oneonly one type of DNA type of DNA sequencesequence......NOT ALWAYS TRUENOT ALWAYS TRUE..