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Gel Electrophoresis of DNA
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Apparatus and Preparation
TABLE OF CONTENTS Apparatus and Preparation Results
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Apparatus and Preparation
Electrophoresis Apparatus Loading Gel Beds
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Electrophoresis Apparatus
chamber casting trays tips pipet chamber cover comb scoop
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Loading Gel Beds pipet loaded well empty well Lane
DNA Solute Migration
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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
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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
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SOLUTE MIGRATION THROUGH THE GEL BED
Dye Mixture Unknown DNA Fragment DNA Markers
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Measurement of Fragment Migration Interpretation of Data
Results DNA Solute Banding Measurement of Fragment Migration Interpretation of Data
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Gel Bed with DNA Solute Banding
Marker Fragment Lane Unknown Fragment Lane continue animation Migration Direction
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Immersion of the gel bed in methylene blue allows the DNA fragments to appear as bands
Unknown DNA Fragment DNA Markers continue animation
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Lighter fragments migrate farther from starting well
Heavier fragments do not migrate as far Unknown DNA Fragment Lighter Fragments Heavier Fragments DNA Markers
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Measurement of Fragment Migration Distances on Gel Bed
Lighter Fragments (Lower Molecular Weight) Heavier Fragments (Higher Molecular Weight) Measure Distances Unknown Fragment continue animation
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Measure distance fragments migrate from starting well (in millimeters) ...
for Unknown DNA Fragment for DNA Markers continue animation
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Fragments migrated (in millimeters) ...
15 for Unknown DNA Fragment 24 18 12 6 5 3 for DNA Markers continue animation
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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 ?
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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
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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
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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)
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