HOME-MADE GENECLEAN KIT

mbxspd at unicorn.nott.ac.uk mbxspd at unicorn.nott.ac.uk
Thu Nov 10 11:20:29 EST 1994


Due to a fair amount of interest, I thought I would post this protocol on the board:

Gene-Clean 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 H2O) and add 5 microlitres to
agarose solution (5 microlitres should be used for up to 5
microgrammes DNA and thereafter an extra 1 microlitre should be used
for each extra microgramme 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 4.5M NaI, 0.1% sodium thiosulphate, re-pellet and
discard supernatant.
	7) Wash pellet 3x in 2 x TE, pH 7.5, 100mM NaCl, 70% EtOH. After last
centrifugation, remove all trace of the supernatant and allow to
air-dry for 5 minutes.
	8) Elute DNA at 65¡C in 25 microlitres TE, pH 8.0 for 5 minutes.
	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.

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 wash 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).

I hope this will help people in some way. Btw, a standard disclaimer:
This works well in our lab bur as it's home-made, I take no 
responsibility if it dosn't work as well as you'd hoped. If you get
a problem with it, drop me a line and maybe we've already had it and
solved it :)

Simon Dawson
Dept. Biochemistry
Queens Medical Centre
Nottingham University
UK

Internet email: mbxspd at unicorn.nott.ac.uk



More information about the Methods mailing list