Team:TU Darmstadt/Protocols/Agarose gel electrophoresis

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Revision as of 14:49, 9 September 2012

Contents

Agarose gel electrophoresis (AGE)

Agarose gel electrophoresis is the most common used method to separate nucleic acids. Due to their negativ charge DNA and RNA molecules can be moved through an agarose gel by an electric field (electrophoresis). Longer molecules move slover through the agarose matrix while short move faster and migrate further.

Procedure

AGE is used for seperation of DNA fragment corresponding to their respective nucleotide lenght

  1. The agarose gel is mixed with 0.0005 vol % of ethidium bromide solution (1mg/µl) alternatively ROTI-safe
  2. A current of 120 V and 50mA is employed

Modifications

  • Fragments longer than 500 bp are separated in 0.8% - 1% agarose-TAE gels
  • Fragments shorter than 500 bp are separated in 2% agarose-TAE gels

Materials

  • TAE-solution (50x batch)
Tris base 242 g
Glacial Acetic Acid 57.1 mL
0.5M EDTA 100 mL
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