Agarose Gel Electrophoresis.ppt i1y1b

Agarose Gel Electrophoresis

Agarose Gel Electrophoresis DNA (-)

• Gel electrophoresis

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Power

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– 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)

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 (1×1 µm)

1% agarose

2% agarose

DNA buffer 

 wells

Cathode (negative)

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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

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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

bromophenol blue

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

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 Ward’s - 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)

wells

DNA ladder 

PCR Product

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

base pairs

DNA ladder

Distance migrated

DNA ladder

base pairs

x bp

sample

Distance migrated

Results

Grp. 7

Grp. 6

Grp. 5

Grp. 4

Grp. 3

Grp. 2

RESULTS

Grp. 1

marker

4 BIO 4

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 marker

Grp. 1

Grp. 2

Grp. 3

Grp. 4

Grp. 5

Grp. 6

Grp. 7

Grp. 8

RESULTS

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

(From product insert of Promega 1 kb DNA ladder)

Grp. 1

RESULTS

marker

4 BIO 5

wells

4 BIO 2 RESULTS marker

Grp. 1

Grp. 2

NONE

Grp. 4

Grp. 5

Grp. 6

Grp. 8

Grp. 3

Grp. 7

(From product insert of Promega 1 kb DNA ladder)

wells

Grp. 7

Grp. 6

Grp. 5

Grp. 4

Grp. 3

Grp. 2

RESULTS

Grp. 1

marker

4 BIO 6

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)