Agarose Purification DNA Fragments

Written by Super User. Posted in DNA

Learn about DNA fragment purification from agarose protocol.

Copyright 2014 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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