1. Excise DNA band from ethidium bromide stained agarose gel (run in TAE).
2. Remember ethidium bromide is a mutagen-wear gloves, lab coat and safety glasses. Read the MSDS before using this material.
3. Always wear eye protection when using the transilluminator as UV light is harmful to your eyes.
4. Weigh gel slice in microfuge tube and add 200 ul/0.1 gm gel of the sodium iodide solution. Sodium Iodide is corrosive- although we use small amounts wear gloves and safety glasses. If you spill some on yourself, wash it off thouroughly. Read the MSDS before using this material.
5. Heat at 50-55°C, 2-5 mins to dissolve agarose, heat longer if not completely dissolved, vortex to mix.
6. Add 2 ul Glassmilk/Prepagene matrix suspension for 1-10 ug DNA (don't use less than 1 ul, and be sure to resuspend the DNA binding matrix THOROUGHLY before using). Yes - we use MUCH less than the amount suggested by the manufacturer. You don't need the amount they suggest. It just makes them more money and actually increases the likely-hood that you will end up with a small amount of matrix in your eluted DNA (bad). Vortex + again after 2-3 min.
7. Place on ice 5 mins (or room temperature). (RT gives better recovery of DNA) note: for small DNA fragments (<500bp)heating the solution to 55°C improves yield- see Biotechniques June 1995, p790.
8. Spin down 1 min in microfuge, aspirate off supernatant.
9. Resuspend pellet in 200 ul Nal solution and spin down again.
10. Aspirate off supernatant, add 200 ul NEW Wash, resuspend and spin down.
11. Repeat once more and remove all excess liquid.
ELUTION
Add 10 ul TE, resuspend, heat 50°C 5 minutes, spin down, remove TE - save.
Repeat once more.
Spin DNA sample prior to use to remove the last few glass grains.
The reagents can be purchased from BIO 101 under the name Geneclean or from BioRad as Prepagene. However, here are recipes for the Nal solution and the Wash. Since we use so much less of the DNA binding matrix it makes sense to prepare extra buffer.
Nal solution:
90.8 gm Nal
1.5 gm Na2SO3 (sulphite!)
dissolve in 100 ml water, filter through 0.45 um filter then add another 0.5 gm sodium sulphite and store at
+4°C.
It is light sensitive so use dark bottles.
Wash solution: 50% ethanol (FLAMMABLE) in: 0.1 M NaCl,
