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Gel Electrophoresis of DNA Quit Gel Electrophoresis of DNA Start.

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Presentation on theme: "Gel Electrophoresis of DNA Quit Gel Electrophoresis of DNA Start."— Presentation transcript:

1 Gel Electrophoresis of DNA Quit Gel Electrophoresis of DNA Start

2 Gel Electrophoresis of DNA Quit Apparatus and Preparation Results TABLE OF CONTENTS

3 Gel Electrophoresis of DNA Quit Electrophoresis Apparatus Loading Gel Beds Apparatus and Preparation

4 Gel Electrophoresis of DNA Quit Electrophoresis Apparatus chamber casting trays comb scoop pipet chamber cover tips

5 Gel Electrophoresis of DNA Quit Loading Gel Beds Lane empty well loaded well pipet DNA Solute Migration

6 Gel Electrophoresis of DNA Quit LOADING THE GEL BED WELL DYE MIXTURE SAMPLE LOADED INTO WELL 2 DNA UNKNOWN SAMPLE LOADED INTO WELL 4 DNA MARKERS SAMPLE LOADED INTO WELL 7 continue animation

7 Gel Electrophoresis of DNA Quit 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. 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 Negatively charged DNA fragments and dye mixture solutes will migrate toward the positive pole. continue animation

8 Gel Electrophoresis of DNA Quit SOLUTE MIGRATION THROUGH THE GEL BED Unknown DNA Fragment Unknown DNA Fragment Dye Mixture DNA Markers

9 Gel Electrophoresis of DNA Quit DNA Solute Banding Measurement of Fragment Migration Measurement of Fragment Migration Results Interpretation of Data

10 Gel Electrophoresis of DNA Quit Gel Bed with DNA Solute Banding Marker Fragment Lane Unknown Fragment Lane Migration Direction continue animation

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

12 Gel Electrophoresis of DNA Quit Lighter fragments migrate farther from starting well Lighter fragments migrate farther from starting well Unknown DNA Fragment Unknown DNA Fragment Heavier fragments do not migrate as far Heavier fragments do not migrate as far DNA Markers Lighter Fragments Heavier Fragments

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

14 Gel Electrophoresis of DNA Quit Measure distance fragments migrate from starting well (in millimeters)... Measure distance fragments migrate from starting well (in millimeters)... for Unknown DNA Fragment for Unknown DNA Fragment for DNA Markers for DNA Markers continue animation continue animation

15 Gel Electrophoresis of DNA Quit Fragments migrated (in millimeters)... Fragments migrated (in millimeters)... for Unknown DNA Fragment for Unknown DNA Fragment for DNA Markers for DNA Markers continue animation continue animation

16 Gel Electrophoresis of DNA Quit DNA MARKER FRAGMENT KNOWN MARKER FRAGMENT LENGTH (in kilobase pairs) DISTANCE FRAGMENT MIGRATED ON GEL BED (in millimeters) UNKNOWN DNA FRAGMENT 15 ? ?

17 Gel Electrophoresis of DNA Quit Fragment Distance Migration (mm) Fragment LengthKBPFragment LengthKBP Migration of fragment is plotted in millimeters (mm) along horizontal axis. Semilog Graph Paper Length of fragment is plotted in kilobase pairs (KBP) along vertical ordinate. continue animation

18 Gel Electrophoresis of DNA Quit Fragment Distance Migration (mm) Plot data points Approximate an area around each point Draw a “best fit” straight line through these areas Fragment LengthKBPFragment LengthKBP Relationship of Fragment Length to Distance Migrated best fit line continue animation

19 Gel Electrophoresis of DNA Quit Fragment Distance Migration (mm) Fragment LengthKBPFragment LengthKBP Relationship of Fragment Length to Distance Migrated 3 5 Draw a vertical line up to meet best fit line Draw a horizontal line over to the ordinate length of unknown fragment is 3.75 KBP Unknown fragment migrated 15 mm best fit line


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