Home-Made Jean-Kleen

Nigel Kenward mbxnjk at vax.nott.ac.uk
Fri Aug 18 02:48:13 EST 1995


Hi There,
This is an excellent method which was adapted from the quoted reference 
by Simon Dawson (department as below). Works as well as Gene-Clean I did 
(we haven't bothered to try Mark II).
Also you could try Biotechniques 18: 970-975 (1995) which suggests that 
increasing the temp of the binding step will improve yields.

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 7.5ml to agarose 	
solution (7.5ml should be used for up to 5mg DNA and thereafter an 	
extra 1ml should be used for each extra mg 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 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 25ml 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. 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).



Good Luck,
Nigel


Nigel Kenward              TEL:+44 (0)115 9249924 ex. 44789
Dept. of Biochemistry      FAX:+44 (0)115 9422225
Queens Medical Centre      Email:mbxnjk at vax.nott.ac.uk
Clifton Boulevard           
Nottingham						
U.K.                   "Back off man, I'm a scientist!"  -- Bill Murray.





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