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Gel Extraction (for double bands)

Promega Wizard Kit

1. Using the small gel rig, pour a 1% low melting point agarose gel (0.5 g in 50mL 1XTBE, 1uL ethidium bromide).

2. Add 5uL of loading dye to your PCR and load the entire volume onto the gel.

3. Run at 70 volts for 1-1.5 hours.

4. Protecting yourself from the UV, and using a razor blade or spatula, excise the band you want and put it in a 1.5mL tube.

5. Weigh each tube and add 10uL of Membrane Binding Solution per 10mg or gel.

6. Place in the 55 C incubator for 10 minutes, or until the gel is dissolved.

7. Transfer entire volume onto minicolumn; let sit 1 minute.

8. Centrifuge at 13000 rpm for 1 minute; discard flow-through.

9. Add 700uL Membrane Wash Solution.

10. Centrifuge at 13000 rpm for 1 minute; discard flow-through.

11. Add 500uL Membrane Wash Solution.

12. Centrifuge at 13000 rpm for 5 minutes; discard flow-through.

13. Re-centrifuge for 1 minute with lid open.

14. Transfer minicolumn to a 1.5mL tube.

15. Add 50uL nuclease-free water (included in kit); let sit 1 minute.

16. Centrifuge at 13000 rpm for 1 minute.

17. Go directly to sequencing or re-amplify using a regular PCR protocol to concentrate product.

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