Presentation is loading. Please wait.

Presentation is loading. Please wait.

DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer.

Similar presentations


Presentation on theme: "DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer."— Presentation transcript:

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 - +

14


Download ppt "DNA QUANTITATION. 2 methods for DNA Quantitation I. Agarose Gel Electrophoresis II. Spectrophotometer."

Similar presentations


Ads by Google