dna quantitation. 2 methods for dna quantitation i. agarose gel electrophoresis ii....
TRANSCRIPT
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
-
+
