Source for glassmilk
mbzspd at granby.nott.ac.uk
Fri Nov 17 03:57:02 EST 1995
Homemade Jean-Kleen protocol (Yes, I know it's a daft name!)
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 H2O or Fluka analytical filter aid
resuspended 1:4 in sterile nanopure H2O) 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 0.5ml 4.5M NaI, 0.1% sodium thiosulphate, re-pellet and
7) Wash pellet 3x in 0.5ml of 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
H2O and final ethanol was made minus ethanol, autoclaved and ethanol
added to a final concentration of 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.
Well, the above works for me every time. Hope it's of some help.
Dr. Simon Dawson TEL:+44 (0)115 9249924 ex. 44789
Dept. of Biochemistry FAX:+44 (0)115 9422225
Queens Medical Centre Email:Simon.Dawson at .nott.ac.uk
Clifton Boulevard http://www.ccc.nottingham.ac.uk/~mbzspd/Simon.html
U.K. "Back off man, I'm a scientist!" - Bill Murray.
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