dna quantitation

Download DNA  QUANTITATION

Post on 01-Jan-2016

23 views

Category:

Documents

1 download

Embed Size (px)

DESCRIPTION

DNA QUANTITATION. 2 methods for DNA Quantitation. I. Agarose Gel Electrophoresis. II. Spectrophotometer. Gel Electrophoresis. - Electrophoresis is a technique used for separation of charged molecules. - PowerPoint PPT Presentation

TRANSCRIPT

  • DNA QUANTITATION

  • 2 methods for DNA QuantitationI. Agarose Gel ElectrophoresisII. Spectrophotometer

  • Gel 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.

  • Agarose Gelextracted from seaweedlinear polymer structure

  • DNA has a negative charge because of the phosphate backboneIt migrates in an electric field from negative charge to positive charged cathodeMobility of DNA Molecules

  • Supercoil DNA migrate faster than circular DNA and linear DNA migrate slowlyI. Conformation of DNA Mobility of DNA MoleculesShape & Size

  • II. Length of DNA fragmentMobility of DNA Molecules

  • Gel ConcentrationHigher concentrations of agarose facilitate separation of small DNALow agarose concentrations allow resolution of larger DNA Mobility of DNA Molecules

  • Gel Concentration

  • Voltage Applied

  • Electrophoresis BufferTAE (Tris-acetate-EDTA) TBE (Tris-borate-EDTA)Loading Buffer6x Loading Dye0.25% Bromophenol blue BB (or tiny amount on the spatula tip)0.25% Xylene cyanol FF XC (or same as BB)15% Ficoll (Type 400)

  • Visualization of DNA FragmentsEthidium Bromide

  • Std.100 ngstd. 300 ngstd. 500 ng std. 1000 ngKDML105-1KDML105-2KDML105-3IR62266-1IR62266-2IR62266-3BT-1BT-2BT-3IR62266(6)Standard of DNA concentrationSamples-+

Recommended

View more >