Download presentation
Presentation is loading. Please wait.
Published byDarleen Melton Modified over 9 years ago
1
DNA QUANTITATION
2
2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer
3
Gel Electrophoresis - Electrophoresis - Electrophoresis is a technique used for separation of charged molecules. - DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose.
4
Agarose Gel extracted from seaweed linear polymer structure
5
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
6
Supercoil DNA Circular form of DNALinear DNA Supercoil DNA migrate faster than circular DNA and linear DNA migrate slowly I. Conformation of DNA Mobility of DNA Molecules Shape & Size
7
II. Length of DNA fragment Mobility of DNA Molecules
8
Gel Concentration Higher concentrations of agarose facilitate separation of small DNA Low agarose concentrations allow resolution of larger DNA Mobility of DNA Molecules
9
Gel Concentration
10
Voltage Applied Cathode (-) Anode (+) As the voltage applied to a gel is in creased, the DNA fragment migrate faster than low voltage.
11
Electrophoresis Buffer TAE (Tris- acetate-EDTA) TBE (Tris-borate- EDTA) Loading Buffer 6x Loading Dye 0.25% Bromophenol blue – BB— (or tiny amount on the spatula tip) 0.25% Xylene cyanol FF –XC— (or same as BB) 15% Ficoll (Type 400)
12
Visualization of DNA Fragments Ethidium Bromide
13
Std.100 ng std. 300 ng std. 500 ng std. 1000 ng KDML105-1 KDML105-2 KDML105-3 IR62266-1 IR62266-2 IR62266-3 BT-1 BT-2 BT-3 IR62266(6) Standard of DNA concentrationSamples - +
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.