Agarose Gel Electrophoresis.ppt

Agarose Gel Agarose Gel ElectrophoresisElectrophoresis

Agarose Gel ElectrophoresisAgarose Gel Electrophoresis

• Gel electrophoresis– separates molecules – different rates of

movement through a gel under the influence of an electrical field( “carrying within electricity”)

– widely used technique for the analysis of:

• nucleic acids (Agarose Gel Electrophoresis)

• Proteins (SDS-PAGE)

+-

Power

+-

Power

DNA (-)DNA (-)

smalllarge

Agarose GelAgarose Gel

• Agarose is a linear polymer derived from red seaweed.

• Malasian word: “agar-agar”.

• Pores = sieve• Increasing agarose

concentration:– decreases pore size– limits the size range of

molecules that can be separated.

Agarose

D-galactose 3,6-anhydroL-galactose

1% agarose 2% agarose

Scanning Electron Micrograph

of Agarose Gel (1×1 µm)

buffer

Cathode(negative)

Anode(positive)

wells

DNA

Sample Preparation and Sample Preparation and LoadingLoading6X Loading Buffer:

Bromophenol Blue (tracking dye) Glycerol/ Glucose/ Sucrose (increase sample density)

DNA MigrationDNA Migration

• Size– migration rate of DNA fragment and logarithm of its

size (in basepairs): linear relationship (inverse)– Larger molecules move more slowly because of more

friction

• Electrical field strength• Buffer (TAE, TBE)• Agarose Gel Concentration• Sample loading

DNA Ladder Standard

Serves as a marker to determine the sizes of unknown DNAs.

100 200 300

1,650

1,000

500

850

650

400

12,000 bp

5,000

2,000

-

+

DNAmigration

bromophenol blue

Visualization

• Ethidium bromide– binds to DNA and fluoresces

under UV light– can be added to the gel and/or

running buffer before electrophoresis or used as developing solution after electrophoresis

– CAUTION: Powerful mutagen and moderately toxic

– Decontamination • Lunn and Sansone Method : + 20

mL 5% hydrophosphorous acid and 12 mL 0. 5 M sodium Nitrate for every 100 mL EtBr (20 hrs)

• Armour method: Bleach (2-3 days)

• Charcoal Filtration

advantagesInexpensiveLess toxicNo UV light requiredNo hazardous waste disposal

disadvantagesLess sensitiveMore DNA needed on gelLonger staining/destaining time

Safer Alternatives to Ethidium Safer Alternatives to Ethidium BromideBromide

• Methylene Blue

• BioRAD - Bio-Safe DNA Stain

• Ward’s - QUIKView DNA Stain

• Carolina BLU Stain

Visualizing the DNA (QuikVIEW stain)Visualizing the DNA (QuikVIEW stain)

250

1,500 1,000

500 750

2,000 bp

DNA ladder

PCRProduct

wells

+ - - - - + + - - + - +

Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA

Distance migrated

base

pai

rs

Determining sample sizeDetermining sample size

• DNA migration rate and logarithm of its size (in basepairs): inverse linear relationship

DNA ladder

Distance migrated

base

pai

rs

x bp

DNA ladder

sample

ResultsResults

wells

10,000 bp

8,000

6,000 5,000 4,000

3,000 2,500

2,000

1,500

1,000

750

ma

rke

r

Grp

. 1

Grp

. 2

Grp

. 3

Grp

. 4

Grp

. 5

Grp

. 6

Grp

. 7

(From product insert of Promega 1 kb DNA ladder)

4 BIO 4 RESULTS

wells

Grp

. 8

Grp

. 6

Grp

. 5

Grp

. 4

Grp

. 2

Grp

. 1

(From product insert of Promega 1 kb DNA ladder)

4 BIO 3 RESULTS m

ark

er

Grp

. 7

Grp

. 3

wells

ma

rke

r

Grp

. 1

Grp

. 2

Grp

. 3

Grp

. 4

Grp

. 5

Grp

. 6

Grp

. 7

(From product insert of Promega 1 kb DNA ladder)

4 BIO 5 RESULTS

SA

MP

LE

1

SA

MP

LE

2

SA

MP

LE

3

wells

(From product insert of Promega 1 kb DNA ladder)

4 BIO 2 RESULTS

Grp

. 7

Grp

. 6

Grp

. 5

Grp

. 4

Grp

. 2

Grp

. 1

ma

rke

r

Grp

. 3

NO

NE

Grp

. 8

wells

10,000 bp

8,000

6,000 5,000 4,000

3,000 2,500

2,000

1,500

1,000

750

ma

rke

r

Grp

. 1

Grp

. 2

Grp

. 3

Grp

. 4

Grp

. 5

Grp

. 6

Grp

. 7

(From product insert of Promega 1 kb DNA ladder)

4 BIO 6 RESULTS