Gene-Kleen protocol

 

Gene-Kleen protocol

Purification of DNA from agarose gels is an essential method involved in the sub-cloning of DNA fragments. The following method describes a variation of the method of Vogelstein and Gillespie, 1979 (Proc. Natl. Acad. Sci. USA. 76, (2) 615-619).

1) Excise the band of interest from the TAE gel using a clean scalpel blade and place in a pre-weighed eppendorf tube.

2) Add 3 volumes of 6M NaI, 0.1% sodium thiosulphate solution and allow agarose to melt (approx. 5 minutes with vortexing). For TBE gels, 0.1 volumes of 1M mannitol should also be added to aid gel solubilisation.

3) Vortex glass suspension (finely crushed glass scintillation vial suspended 1:1 in sterile nanopure H₂ O or Fluka analytical filter aid resuspended 1:4 in sterile nanopure H₂ O) and add 7.5ul to agarose solution (7.5ul should be used for up to 5ug DNA and thereafter an extra 1ul should be used for each extra ug DNA).

4) Allow DNA to bind for 15-20 minutes at room temperature.

5) Spin down glass-DNA for 30 secs. in a microfuge at maximum speed and carefully remove supernatant. Discard supernatant.

6) Wash pellet in 500ul 4.5M NaI, 0.1% sodium thiosulphate, re-pellet and discard supernatant.

7) Wash pellet 2 x 500ul in 1 x TE, pH 7.5, 200mM NaCl, 60% EtOH. After last centrifugation, remove all trace of the supernatant and allow to air-dry for 5 minutes.

8) Elute DNA at 37 - 55°C in 25ul TE, pH 8.0 for 5 minutes.

N.B: It is important to elute in a buffer with a pH of ca. 8 as elution in water or lower pH buffers decreases the yield markedly.

9) Spin down glass for 5 minutes at maximum speed in a microfuge and carefully remove supernatant to a clean eppendorf tube.

10) Repeat elution step and pool supernatants. Discard pellet. Respin pooled sample to remove traces of glass for 5 mins and transfer clean supernatant to a fresh, sterile eppendorf tube.

All reagents are made from the highest grade chemicals available (esp. important for the NaI). NaI solutions were made with sterile, nano-pure H₂ O and final ethanol was made minus ethanol, autoclaved and ethanol added to a final concentration o f 70% (v/v) after sterilisation. Glass 'beads' were made from a finely crushed scintillation vial (i.e high quality glass) by crushing with a pestle and mortar. Glass is crushed basically until your wrist feels like it's about to fall off...and then some (should behave like cooking flour). Alternatively, I have recently tried Celite Analytical Filter Aid (Celite AFA, Cat. No. 22137 - I think thats the catalogue number anyhow!) from Fluka in place of the crushed glass with brilliant results.