Summary: Isolation of DNA From Agarose Gel With Silica
1. Weigh gel slice in 1.5 ml tubes (maximum = 0.5 g). Add 2 µl of NaI solution per mg of gel slice.
For PCR or digestion clean up, add 1/20 volume 1 M NaPi (or KPi) pH 6. Optimum binding to
silica is achieved at pH 6.5. Add 2 µl of NaI solution per µl of reaction volume. Go to step 3.
2. Incubate at 37-50°C until gel slice dissolves (usually 20-30 minutes at 50°C for ~1% agarose).
3. Add 1 µl of silica slurry per 1 µg of DNA (estimated). Immediately mix by shaking tube.
NB: Before use, thaw and vigorously vortex the silica slurry suspension for >1 min.
4. Place on rotator at 4°C for 15-30 minutes.
5. Spin for 10 minutes at top speed using microfuge with swing-out rotor (10,000 rpm; 11,700 g).
6. Wash pellet 2X with Wash Buffer. Disperse silica pellet by "ripping" tube across tube rack.
Centrifuge as described above for 5 minutes. Spin briefly and remove residual Wash Buffer.
7. Resuspend silica in 10-20 µl TE pH 8 by repipetting. Alternatively, use 10 mM Tris pH 8.5.
8. Heat 10 minutes at 37°C (or 5 minutes at 50°C). Centrifuge for 10 minutes at top speed using
microfuge with swing-out rotor. Transfer supernatant containing DNA to a fresh tube.
NB: repeat elution with dH2O to capture remaining 10-20% DNA and concentrate in Speedvac.
Sodium Iodide Solution (6M):
Dissolve 9.1 g NaI in 10 ml dH2O (freshly prepare each time, 149.9 g/mol).
1:1 to 4:1 100% EtOH:TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8)
Current Protocols in Molecular Biology suggests 4:1 ratio (80% EtOH).