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Agarose Gel Electrophoresis

Agarose Gel Electrophoresis


DNA (-) small large

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

Agarose Gel
Agarose Agarose is a linear polymer derived from red seaweed. Malasian word: agaragar. Pores = sieve Increasing agarose concentration:
decreases pore size limits the size range of molecules that can be separated.
D-galactose 3,6-anhydro L-galactose

Scanning Electron Micrograph of Agarose Gel (11 m)

1% agarose

2% agarose

DNA buffer wells

Cathode (negative)

Anode (positive)

Sample Preparation and Loading


6X Loading Buffer: Bromophenol Blue (tracking dye) Glycerol/ Glucose/ Sucrose (increase sample density)

DNA 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.
12,000 bp 5,000

DNA migration

2,000 1,650 1,000 850 650 500 400 300 200 100

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

Safer Alternatives to Ethidium Bromide Methylene Blue BioRAD - Bio-Safe DNA Stain Wards - QUIKView DNA Stain Carolina BLU Stain
advantages
Inexpensive Less toxic No UV light required No hazardous waste disposal

disadvantages
Less sensitive More DNA needed on gel Longer staining/destaining time

Visualizing the DNA (QuikVIEW stain)


DNA ladder

wells

PCR Product

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

2,000 bp 1,500 1,000 750 500 250

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

Determining sample size

DNA ladder

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

base pairs

Distance migrated

DNA ladder

x bp base pairs
sample

Distance migrated

Results

marker

Grp. 1

Grp. 2

Grp. 3

Grp. 4

Grp. 5

Grp. 6

Grp. 7

4 BIO 4
RESULTS

wells

10,000 bp 8,000 6,000 5,000 4,000 3,000 2,500 2,000 1,500 1,000 750
(From product insert of Promega 1 kb DNA ladder)

4 BIO 3
RESULTS
Grp. 8 Grp. 7 Grp. 6 Grp. 5 Grp. 4 Grp. 3 Grp. 2 Grp. 1 marker

wells

(From product insert of Promega 1 kb DNA ladder)

SAMPLE 3
SAMPLE 2 SAMPLE 1 Grp. 7 Grp. 6 Grp. 5 Grp. 4 Grp. 3 Grp. 2 Grp. 1 marker

4 BIO 5

RESULTS

wells

(From product insert of Promega 1 kb DNA ladder)

4 BIO 2
RESULTS
Grp. 7 Grp. 3 Grp. 8 Grp. 6 Grp. 5 Grp. 4 NONE Grp. 2 Grp. 1 marker

wells

(From product insert of Promega 1 kb DNA ladder)

marker

Grp. 1

Grp. 2

Grp. 3

Grp. 4

Grp. 5

Grp. 6

Grp. 7

4 BIO 6
RESULTS

wells

10,000 bp 8,000 6,000 5,000 4,000 3,000 2,500 2,000 1,500 1,000 750
(From product insert of Promega 1 kb DNA ladder)

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