Copyright 2014 Molecular Station
DNA Fragment Purification from Agarose Protocol
- Run DNA on "Low melt" agarose gel.
- 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".
- After bands have separated, visualize the band on a UV box (minimize exposure of DNA to UV)
- Cut band out (DO NOT scratch the UV filter on the light box!).
- 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.
- Microfuge 5 mins, remove supernant
- Add 100 l of T.E. to phenol, vortex, heat 65°C 3 min. vortex
- Microfuge, pool supernants.
- Chloroform extract (approx. 400 ul), microfuge 3 min.
- EtOH precipitate, adding 1/10 vol. 3M Na0Ac, 2.5 vol. EtOH.
- -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.