Gel Electrophoresis of DNA

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Presentation transcript:

Gel Electrophoresis of DNA Start

Apparatus and Preparation TABLE OF CONTENTS Apparatus and Preparation Results

Apparatus and Preparation Electrophoresis Apparatus Loading Gel Beds

Electrophoresis Apparatus chamber casting trays tips pipet chamber cover comb scoop

Loading Gel Beds pipet loaded well empty well Lane DNA Solute Migration

LOADING THE GEL BED DYE MIXTURE SAMPLE LOADED INTO WELL 2 1 DYE MIXTURE SAMPLE LOADED INTO WELL 2 2 3 DNA UNKNOWN SAMPLE LOADED INTO WELL 4 4 5 6 DNA MARKERS SAMPLE LOADED INTO WELL 7 7 8 continue animation

Gel bed is submerged in a buffer and an electrical current is placed across the chamber. Solutes are pulled through the gel bed toward the pole opposite to their charge. - + WELL 1 Negatively charged DNA fragments and dye mixture solutes will migrate toward the positive pole. 2 3 4 5 6 7 8 continue animation

SOLUTE MIGRATION THROUGH THE GEL BED Dye Mixture Unknown DNA Fragment DNA Markers

Measurement of Fragment Migration Interpretation of Data Results DNA Solute Banding Measurement of Fragment Migration Interpretation of Data

Gel Bed with DNA Solute Banding Marker Fragment Lane Unknown Fragment Lane continue animation Migration Direction

Immersion of the gel bed in methylene blue allows the DNA fragments to appear as bands Unknown DNA Fragment DNA Markers continue animation

Lighter fragments migrate farther from starting well Heavier fragments do not migrate as far Unknown DNA Fragment Lighter Fragments Heavier Fragments DNA Markers

Measurement of Fragment Migration Distances on Gel Bed Lighter Fragments (Lower Molecular Weight) Heavier Fragments (Higher Molecular Weight) Measure Distances Unknown Fragment continue animation

Measure distance fragments migrate from starting well (in millimeters) ... for Unknown DNA Fragment for DNA Markers continue animation

Fragments migrated (in millimeters) ... 15 for Unknown DNA Fragment 24 18 12 6 5 3 for DNA Markers continue animation

KNOWN MARKER FRAGMENT LENGTH (in kilobase pairs) DISTANCE FRAGMENT MIGRATED ON GEL BED (in millimeters) DNA MARKER FRAGMENT 9.0 3 1 7.0 2 5 3 6.5 6 5.0 12 4 3.0 18 5 2.0 6 24 UNKNOWN DNA FRAGMENT 15 ?

Fragment Distance Migration (mm) Semilog Graph Paper 10 Length of fragment is plotted in kilobase pairs (KBP) along vertical ordinate. 9 Fragment Length KBP 8 7 6 5 4 3 Migration of fragment is plotted in millimeters (mm) along horizontal axis. 2 1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Fragment Distance Migration (mm) continue animation

Relationship of Fragment Length to Distance Migrated 10 9 Fragment Length KBP Plot data points 8 7 Approximate an area around each point 6 Draw a “best fit” straight line through these areas 5 4 best fit line 3 2 1 2 3 4 5 6 8 10 12 14 16 18 20 22 24 26 28 Fragment Distance Migration (mm) continue animation

Relationship of Fragment Length to Distance Migrated 10 9 Fragment Length KBP 8 Draw a vertical line up to meet best fit line 7 Draw a horizontal line over to the ordinate 6 5 4 best fit line length of unknown fragment is 3.75 KBP 3 2 Unknown fragment migrated 15 mm 1 2 3 4 5 6 8 10 12 14 16 18 20 22 24 26 28 Fragment Distance Migration (mm)