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1. Oligos or small DNA fragments may be separated on
a PAGE or PAGE/urea gel (e.g., 20% polyacriamide/8M urea in TBE buffer).
See also Effective range of separation of DNAs in PAGE
and
Effective range of separation of DNAs in PAGE/Urea.
2. Visualize DNA by UV shadowing or staining.
3. Cut out the desired band and slice it into small pieces.
4. Transfer the gel slices to a microcentrifuge tube.
5. Add 2 volumes of TE or ddH2O.
6. Incubate overnight with gentle rocking.
7. Centrifuge to pellet acrylamide.
8. Transfer the supernatant to a fresh tube.
9. Wash the acrylamide pellet with 1 volume of TE or ddH2O.
10. Centrifuge and pool the supernatant.
Precipitation
11. If stained with ethidium bromide, extract once with n-butanol.
12. Extract once with phenol:chloroform:isoamyl alchol (25:24:1).
13. Add sodium acetate to the final concentration of
0.5 M. Precipitate with 2 volumes of ethanol at -70 °C for > 1 hour.
14. Centrifuge at 4 °C for 15 minutes to pellet DNA.
15. Wash the pellet with cold 70% ethanol.
16. Air-dry.
17. Resuspend DNA in 20-50 ml of
TE or ddH2O.
Desalting
11a. Desalting with a desalting column.
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