Wittrup: Gel extraction

Materials

QIAquick Gel Extraction kit (Qiagen 28706)

42-50˚C water bath

Protocol

This protocol is adapted/condensed from the Qiagen instructions. The Qiagen site ([1]) offers several helpful tips that are useful for first-time users and for troubleshooting.

1. Using a razor blade, cut the desired piece from the gel and place in a microcentrifuge tube.

2. Add 3 gel volumes (often 300 μL for a small slice) of Buffer QG to the vial to dissolve agarose.

3. Incubate at 42-50˚C for 10 min. or until gel is fully dissolved. Twice vortex briefly.

4. Upon complete dissolution, verify proper pH by checking for yellow color.

5. [Optional: only beneficial if DNA is <500 bp or >4000 bp] Add 1 gel volume of isopropanol to the vial and mix gently; do not centrifuge.

6. Apply sample to QIAquick column in a collection tube.

7. Centrifuge at 15,000-18,000g for 60 s and discard flowthrough.

8. Add 500 μL of Buffer QG to column to dissolve residual agarose.

9. Centrifuge at 15,000-18,000g for 60 s and discard flowthrough.

10. Add 750 μL of wash Buffer PE to column.

11. Centrifuge at 15,000-18,000g for 60 s and discard flowthrough.

12. Centrifuge at 15,000-18,000g for 60 s and discard flowthrough (to eliminate residual ethanol).

13. Place column in vial.

14. Add 30 μL of elution Buffer EB to center of column.

15. Let stand for 60 s.

16. Centrifuge at 15,000-18,000g for 60 s.

17. Store DNA collected in vial at -20˚C.