PLASMID ISOLATION AND ANALYSIS Part IV DNA Analysis Using Agarose Gel Electrophoresis
DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II....
-
Upload
darleen-melton -
Category
Documents
-
view
274 -
download
1
Transcript of DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II....
![Page 1: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/1.jpg)
DNA QUANTITATION
![Page 2: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/2.jpg)
2methods for DNA Quantitation
I. Agarose Gel Electrophoresis
II. Spectrophotometer
![Page 3: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/3.jpg)
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. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/4.jpg)
Agarose Gel
extracted from seaweed
linear polymer structure
![Page 5: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/5.jpg)
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. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/6.jpg)
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
![Page 7: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/7.jpg)
II. Length of DNA fragment
Mobility of DNA Molecules
![Page 8: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/8.jpg)
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
![Page 9: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/9.jpg)
Gel Concentration
![Page 10: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/10.jpg)
Voltage Applied
Cathode (-)
Anode (+)
As the voltage applied to a gel is in creased, the DNA fragment migrate faster than low voltage.
![Page 11: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/11.jpg)
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
![Page 12: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/12.jpg)
Visualization of DNA Fragments
Ethidium Bromide
![Page 13: DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.](https://reader031.fdocuments.net/reader031/viewer/2022031716/56649e0c5503460f94af5d07/html5/thumbnails/13.jpg)
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
-
+