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Star Republic: Guide for Biologists

Purification of DNA from agarose gels (Qiagen spin column)

1. Excise the DNA fragment from the agarose gel with a clean scalpel. Remove extra agarose.

2. Transfer gel slice to a colorless tube. Add 3-6 volumes of Buffer QC to 1 volume of gel.

3. Incubate at 50 C for 10 minutes. Mix by vortexing every 2-3 minutes during the incubation.

4. After the gel slice has dissolved completely, check that the color of the mixture is yellow. If the color is orange or violet, pH is too high for DNA binding. Add 10 ml of 3 M sodium acetate (pH 5.2) and mix.

5. Add 1 gel volume of isopropanol to the sample and mix by inverting the tube.

6. Place a MinElute column in a 2 ml collection tube.

7. Apply the sample to the MinElute column and centrifuge for 1 minute.

8. Discard the flow-through.

9. Add 0.5 ml Buffer QG to the MinElute column and centrifuge for 1 minute.

10. Discard the flow-through.

11. Add 0.75 ml of Buffer PE to the MinElute column and centrifuge for 1 minute.

12. Discard the flow-through and centrifuge the MinElute column for an additional 1 minute at maximum speed in a microfuge.

13. Place the MinElute column into a clean 1.5 ml microcentrifuge tube.

14. To elute DNA, add 10 ml of Buffer EB (10 mM Tris-HCl, pH8.5) or TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), or ddH2O, let the column stand for 1 minute, and then centrifuge for 1 minute.