DNA gel purification without a spin column - (Feb/01/2008 )
A past lab member that I had worked with (and since have lost contact) told me that there is a way of cutting a DNA band from agarose, wrapping it in some kind loose woven cloth, placing it in an Epindorf and spinning it at top speed so that the DNA band and agarose separates. He then did an DNA prec, EtOH wash, and resuspension of sample. Has anyone ever heard of this or knows where I might find a protocol.
I think I have answered similar questions somewhere on this board, but it's really simple. You run the low melting temp agarose gel in TAE buffer, cut out the band, melt the gel at 70*C, put it in -80*C for 10-15min, take it out and spin at top speed for 5min, the DNA is in the supernatant, use it directly for ligation.
Sounds like the crush gel method.
Basically the idea is to crush the agarose gel slab in TE, then removing the bits agarose.
Freezing the agarose gel slab at -20 Celsius. Then place the gel slab into a microcentrifuge tube with a tinny hole at its base. Place a second tube under the first tube (which contains the agarose slab) to collect the DNA solution eluted out. You may place both tube into a 15ml Fulcon tube to keep the arrangement steady. And finally centrifuge hard. You should be able to elute out a solution containing your DNA. The agarose is retained in the first tube (which has a hole pricked at its base).
other alternatives include using agarase.... my supervisor used to use that enzyme prior to column.
electrolution.... mainly use when gel purifiying real large DNA fragments... from 12kb to 200Mb.
cut yourself a small square of dialysis membrane and filter paper. Cut a small slit beneath the DNA band you want. Insert filter/membrane (filter must be facing the DNA) and run the band into the filter. Put the filter paper in an 0.5ml eppie with a hole in the bottom; place this in a 1.5ml eppie and spin out your DNA. I routinely do this for cloning, without EtOH ppt or anything to clean up.
thanks everyone for you help