DNA Gel excision protocol

Most gel excisions stink.  Qiagen and Promega tend to claim 80%+ efficiencies that aren’t really achievable.  Here I’m describing Jen Sheen’s SiO2 method for DNA purification from agarose: http://www.plantmethods.com/content/pdf/1746-4811-6-1.pdf

prepare a 0.6% gel to isolate my cassette of interest.

add  6x volumes of 4.5 M Gua isothiocyanate.  If you have NaI or GuHCl should be equivalent.

melt the gel slab(s) for ~ 12 minutes at 60oC.

once your gel/DNA mixture is molten add 20 uL of 300 mg/mL SIO2. –> SiO2 is made by making 3 g of SiO2 in clean water.

incubate this mixture 5 min at room temperature.  Your DNA will bind to the silica and renature nicely.  Mix it well by inversion

spin down 10sec at 16000g.

Critical: You need to do more Ethanol washes to get rid of the isothiocyanate.

add 500 uL of 70% ethanol or PE or AW or whatever comes with your kit that is equivalent.

Vortex.  Vortex well to break up the clump of silica.  This should be OK below 3000 bp DNA fragments.

spin at 16000 g and get rid of the ethanol.  Do this 2 more times.

after the third ethanol removal spin again for 10 seconds at 16000g.  This will compact the pellet and you can pipette off the remaining ethanol with a p200.

Finally resuspended in 20 uL dH2O. (you can probably get away with as little as 5 or 7 uL).  Vortex to loosen the pellet.  Sit your water/silica mixture at 60oC for about 5 min.

Spin again at 16000g and collect your DNA.

Here are some Nanodrop results.  The only thing to take away from these numbers is a nice curve was available.  I normally have ugly curves with column+gel excisions.  Also this is about 20x more efficient than column+gel excisions I normally perform.

260/280; 260/230; ng / uL

1.71; 1.97; 105.9