DNA QUANTITATION

2methods for DNA Quantitation

I. Agarose Gel Electrophoresis

II. Spectrophotometer

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.

Agarose Gel

extracted from seaweed

linear polymer structure

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

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

II. Length of DNA fragment

Mobility of DNA Molecules

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

Gel Concentration

Voltage Applied

Cathode (-)

Anode (+)

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

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

Visualization of DNA Fragments

Ethidium Bromide

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

-

+

http://www-unix.oit.umass.edu/~ansc311/agarose_gel.swf