User:Andrew Perry/Protocols/DNA gel extraction
DNA gel extraction on the cheap
These are some DIY DNA gel extraction methods I tried a few years back. My experience was that none of them were as efficient as the commercial kits (eg Qiagen QIAEX II or Promega Wizard kits), but they may be a starting point for further optimization.
Fast Flush Method (based on Grey & Brendel, 1992)
1. Cut out your band from the gel and trim off any excess agarose.
2. Place the gel slice in the top of a 100 ul filter pipette tip (some filters prevent any moisture passing through them, so don't use this type - most filter tips should be OK). Alternatively, punch a hole in the bottom of a 600 uL microfuge tube and plug with a ball of glass wool (WARNING: don't touch the glass wool without gloves - tiny glass splinters are not fun !).
3. Cut off the bottom of the tip so it fits into a 1.5 ml Eppendorf tube (and doesn't collapse during Step 4).
4. Put the tip into the tube and centrifuge at 14,000 rpm for 30 seconds.
5. Remove the tip and voila - your DNA is in the gel buffer at the bottom of the tube. The agarose is blocked by the filter and stays at the top.
Remember that your DNA will now be in whatever buffer you make your gels with. Yield is fairly good, but I've never done this with DNA fragments larger than 1.5 kb. The whole process takes about 5 minutes and is cheap (1 tip, 1 tube, 1 scalpel blade).
Freeze and Squeeze Method (based on Thuring, 1975; Diethard & Renz 1983)
1. Cut agarose gel slice containing your PCR product (or DNA fragment). Transfer the slice to a 1.5 ml tube. Add 50 ul TE buffer into it.
2. Freeze in liquid N2 (or at -80C, >1 hr).
3. Spin at max speed for 10 to 20 min. Transfer supernatant to new 1.5 ml tube.
4. Add another 50ul TE buffer to the gel pellet. Resuspend the agarose gel. Repeat freezing step, and spin at max. speed for another 10 - 20 min. Pool the supernatant in 1.5 ml tube. [Normally, I'd just repeat the freezing and squeezing (centrifugation) once. You can repeat a few times to get more DNA].
5. Precipitate the pooled supernatant containing DNA using standard protocol (Sambrook et al). Note: Some suggested adding glycogen as DNA carrier, but I don't.
1. Make sure you're not using smaller tubes, i.e. 0.5 ml tube. They can't stand the pressure and will crack, leaking the content out during centrifugation. That's why I use a sturdy 1.5 ml tube.
I've used the purified DNA from above method to do sequencing and cloning without problem.
Roll your own QIAEX II extraction kit:
3M NaI (sodium iodide),
4M NaClO4 (sodium perchlorate),
5mM Tris/HCl, pH7.5 0.1% Na2SO3 (disodium sulphate).
(from http://www.bio.net/bionet/mm/methods/1995-December/037584.html and 'The Qiagenologist' 3rd Edition, 1990.