Gel Electrophoresis of DNA
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Transcript of Gel Electrophoresis of DNA
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Gel Electrophoresis of DNAGel Electrophoresis of DNA
Start
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Apparatus and PreparationApparatus and Preparation
ResultsResults
TABLE OF CONTENTSTABLE OF CONTENTS
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Electrophoresis ApparatusElectrophoresis Apparatus
Loading Gel BedsLoading Gel Beds
Apparatus and PreparationApparatus and Preparation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Electrophoresis ApparatusElectrophoresis Apparatus
chamber
casting trays
combscoop
pipetchamber cover
tips
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Loading Gel BedsLoading Gel Beds
Lane
empty well
loaded well
pipet
DNA Solute Migration
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
LOADING THE GEL BEDLOADING THE GEL BED
WELL
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5
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7
8
3
2
1
DYE MIXTURE SAMPLE LOADED INTO WELL 2
DNA UNKNOWN SAMPLE LOADED INTO WELL 4
DNA MARKERS SAMPLE LOADED INTO WELL 7
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animation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
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
4
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3
2
1+ -
Negatively charged DNA fragments and dye mixture solutes will migrate toward
the positive pole.
continue animationcontinue
animation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
SOLUTE MIGRATION THROUGH THE GEL BEDSOLUTE MIGRATION THROUGH THE GEL BED
Unknown DNAFragment
Unknown DNAFragment
Dye MixtureDye Mixture
DNA MarkersDNA Markers
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
DNA Solute Banding DNA Solute Banding
Measurement of Fragment Migration
Measurement of Fragment Migration
ResultsResults
Interpretation of DataInterpretation of Data
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Gel Bed with DNA Solute BandingGel Bed with DNA Solute Banding
Marker Fragment Lane
Unknown Fragment Lane
Migration Directioncontinue
animationcontinue
animation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Immersion of the gel bed in methylene blue allows the DNA fragments to appear as bands
Immersion of the gel bed in methylene blue allows the DNA fragments to appear as bands
Unknown DNAFragment
Unknown DNAFragment
DNA MarkersDNA Markers
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animation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Lighter fragments migrate farther from starting wellLighter fragments migrate farther from starting well
Unknown DNAFragment
Unknown DNAFragment
Heavier fragments do not migrate as farHeavier fragments do not migrate as far
DNA MarkersDNA Markers
Lighter Fragments
Lighter Fragments
Heavier Fragments
Heavier Fragments
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Measurement of Fragment Migration Distances on Gel Bed
Measurement of Fragment Migration Distances on Gel Bed
MeasureDistances
Lighter Fragments(Lower Molecular Weight)
Heavier Fragments(Higher Molecular Weight)
UnknownFragment
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Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
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 animationcontinue animation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
Fragments migrated (in millimeters) ...Fragments migrated (in millimeters) ...
for Unknown DNA
Fragment
for Unknown DNA
Fragment
for DNA Markers
for DNA Markers
1515
2424 1818 1212 66 55 33
continue animationcontinue animation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
DNA MARKER FRAGMENT
DNA MARKER FRAGMENT
KNOWN MARKERFRAGMENT LENGTH
(in kilobase pairs)
KNOWN MARKERFRAGMENT LENGTH
(in kilobase pairs)
DISTANCE FRAGMENT MIGRATED ON GEL BED
(in millimeters)
DISTANCE FRAGMENT MIGRATED ON GEL BED
(in millimeters)
66
55
44
22
11
33
7.07.0
9.09.0 33
6.56.5
5.05.0
3.03.0
2.02.0
55
66
1212
1818
2424
UNKNOWN DNA FRAGMENT
UNKNOWN DNA FRAGMENT
1515??
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
2 4 6 108 16 18 20 22 24 26 281412Fragment Distance Migration (mm)
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4
1
7
10
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5
Fragment
Length
KBP
Migration of fragment is plotted in millimeters (mm)
along horizontal axis.
Semilog Graph PaperSemilog Graph Paper
Length of fragment is plotted in
kilobase pairs (KBP) along
vertical ordinate.
continue animationcontinue animation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
2 4 6 108 16 18 20 22 24 26 281412Fragment Distance Migration (mm)
2
3
4
1
7
10
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3 5
Plot data points
Approximate an area around each point
Draw a “best fit” straight line through
these areas
Fragment
Length
KBP
Relationship of Fragment Length to Distance Migrated
Relationship of Fragment Length to Distance Migrated
best fit linebest fit line
continue animationcontinue animation
Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit
2 4 6 108 16 18 20 22 24 26 281412Fragment Distance Migration (mm)
2
3
4
1
7
10
9
8
6
5
Fragment
Length
KBP
Relationship of Fragment Length to Distance Migrated
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 unknownfragment is 3.75 KBP
length of unknownfragment is 3.75 KBP
Unknown fragment migrated 15 mm
Unknown fragment migrated 15 mm
best fit linebest fit line