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Agarose Purification DNA Fragments

Copyright 2012 Molecular Station

DNA Fragment Purification from Agarose Protocol

  1. Run DNA on "Low melt" agarose gel.
  2. For 600bp DNA fragments to 5kb use1%; For 300- 700bp use 2% agarose. If you need to run smaller fragments use 2% "Nusieve" + 1% "Low melt".
  3. After bands have separated, visualize the band on a UV box (minimize exposure of DNA to UV)
  4. Cut band out (DO NOT scratch the UV filter on the light box!).
  5. Add 100 ul of T.E. buffer (10mM Tris-HCl pH 7.6, 1mM EDTA) to band, crush, heat to 65oC for approx. 5 min, add 200l of phenol, vortex, heat 65°C for 3 min., vortex.
  6. Microfuge 5 mins, remove supernant
  7. Add 100 l of T.E. to phenol, vortex, heat 65°C 3 min. vortex
  8. Microfuge, pool supernants.
  9. Chloroform extract (approx. 400 ul), microfuge 3 min.
  10. EtOH precipitate, adding 1/10 vol. 3M Na0Ac, 2.5 vol. EtOH.
  11. -20°C 1-2+hrs; spin 30 min., dry down, bring up in suitable vol 0.1X T.E.


Tips for DNA Agarose Gel Purification

  • Set the trans-illuminator to long UV wavelength (or the low-power). This is a great way to minimise the amount of time and energy of UV DNA exposure. This will minimize the UV mutagenesis of the DNA.
  • Trim off as much of agarose from the band as possible without removing DNA. This helps in minimizing agarose contamination which will enhance DNA purification.
  • If you are getting problems use commercial kits such as spin-columns.There are some excellent kits for extracting DNA if you can afford them. These include kits from Qiagen, Sigma, and other companies.

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