Gel Electrophoresis of DNA Quit Gel Electrophoresis of DNA Start.

Gel Electrophoresis of DNAGel Electrophoresis of DNAQuit

Gel Electrophoresis of DNAGel Electrophoresis of DNA

Start


Apparatus and PreparationApparatus and Preparation

ResultsResults

TABLE OF CONTENTSTABLE OF CONTENTS


Electrophoresis ApparatusElectrophoresis Apparatus

Loading Gel BedsLoading Gel Beds

Apparatus and PreparationApparatus and Preparation


Electrophoresis ApparatusElectrophoresis Apparatus

chamber

casting trays

combscoop

pipetchamber cover

tips


Loading Gel BedsLoading Gel Beds

Lane

empty well

loaded well

pipet

DNA Solute Migration


LOADING THE GEL BEDLOADING THE GEL BED

WELL

4

5

6

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

continue animationcontinue

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.

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

5

6

7

8

3

2

1+ -

Negatively charged DNA fragments and dye mixture solutes will migrate toward

the positive pole.


animation


SOLUTE MIGRATION THROUGH THE GEL BEDSOLUTE MIGRATION THROUGH THE GEL BED

Unknown DNAFragment

Unknown DNAFragment

Dye MixtureDye Mixture

DNA MarkersDNA Markers


DNA Solute Banding DNA Solute Banding

Measurement of Fragment Migration

Measurement of Fragment Migration

ResultsResults

Interpretation of DataInterpretation of Data


Gel Bed with DNA Solute BandingGel Bed with DNA Solute Banding

Marker Fragment Lane

Unknown Fragment Lane

Migration Directioncontinue

animationcontinue

animation


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



animation


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


Lighter Fragments

Lighter Fragments

Heavier Fragments

Heavier Fragments


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

continue animationcontinue animation


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



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



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??


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

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.




2

3

4

1

7

10

9

8

6

5

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


best fit linebest fit line




2

3

4

1

7

10

9

8

6

5

Fragment

Length

KBP



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

Gel Electrophoresis of DNA Quit Gel Electrophoresis of DNA Start.

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