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Star Republic: Guide for Biologists

Purification of DNA from PAGE gels

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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.