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Page 1: DNA  QUANTITATION

DNA QUANTITATION

Page 2: DNA  QUANTITATION

2methods for DNA Quantitation

I. Agarose Gel Electrophoresis

II. Spectrophotometer

Page 3: DNA  QUANTITATION

Gel Electrophoresis

- Electrophoresis- Electrophoresis is a techniq ue used for separation of cha

rged molecules.

- DNA is a negatively charged molecule, and is moved by el

ectric current through a matr ix of agarose.

Page 4: DNA  QUANTITATION

Agarose Gel

extracted from seaweed

linear polymer structure

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DNA has a negative charge because of the phosphate backbone

It migrates in an electric field

from negative charge to

positive charged cathode

Mobility of DNA Molecules

Page 6: DNA  QUANTITATION

Supercoil DNA Circular form of DNA Linear DNA

Supercoil DNA migrate faster than circular DNA a

nd linear DNA migrate slowly

I. Conformation of DNA

Mobility of DNA Molecules

Shape & Size

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II. Length of DNA fragment

Mobility of DNA Molecules

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Gel Concentration

Higher concentrations of agarose facilita

te separation of small DNA

Low agarose concentrations allow resoluti on of larger DNA

Mobility of DNA Molecules

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Gel Concentration

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Voltage Applied

Cathode (-)

Anode (+)

As the voltage applied to a gel is in creased, the DNA fragment migrate faster than low voltage.

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Electrophoresis Buffer

-TAE (Tris aceta- te EDTA)

-TBE (Tris borat-e EDTA)Loading Buffer

6x Loading Dye 025. % Bromophenol blue – BB— (or

aa aaaa aa aaa aaaaaaa aaaa) 025. % Xylene cyanol FF –XC— (or sa

aa aaa) 1 5 % ( 4 0 0 )Ficoll Type

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Visualization of DNA Fragments

Ethidium Bromide

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100

Std

.ng

a

a.

30

0

500

std.

ng

1000

std.

n

a

-1051

KDML

-1052

-1053

KD

ML

-622661

IR

-622662

-62266

3 -1BT -a2 -a3 62266

6

Standard of DNA concentration Samples

-

+


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