Site-Directed Mutagenesis Kit


QIAEX II® Gel Extraction Kit

The QIAEX II Gel Extraction Kit (cat. nos. 20021 and 20051) can be stored at room temperature (15–25°C) for up to 12 months. For more information, please refer to the QIAEX II Handbook, which can be found at For technical assistance, please call toll-free 00800-22-44-6000, or find regional phone numbers at

Notes before starting This protocol is for cleanup of DNA fragments of 40 bp to 50 kb. The yellow color of Buffer QX1 indicates a pH ≤7.5. Add ethanol (96–100%) to Buffer PE concentrate before use (see bottle label for volume). A heating block or water bath at 50°C is required. All centrifugation steps are carried out at 17,900 x g (~13,000 rpm) in a conventional tabletop microcentrifuge at room temperature (15–25°C). For purification of DNA from polyacrylamide gels or aqueous solutions, see the handbook.

1. Excise the DNA band from the agarose gel with a clean, sharp scalpel. Use a 1.5 ml microfuge tube for processing up to 250 mg agarose per tube.

2. Weigh the gel slice in a colorless tube. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100 bp; 3 volumes for 100 bp – 4 kb; 3 volumes with 2 volumes of water for >4 kb. Add 6 volumes of Buffer QX1 when using >2% or Metaphor agarose gels.

3. Resuspend QIAEX II by vortexing for 30 s. Add QIAEX II to the sample and mix: Use 10 μl QIAEXII for ≤2 μg DNA; 30 μl for 2–10 μg DNA; and an additional 30 μl for each additional 10 μg DNA. January 2011 Quick-StartProtocol Sample & Assay Technologies For up-to-date licensing information and productspecific disclaimers, see the respective QIAGEN® kit handbook or user manual. Trademarks: QIAGEN®, QIAEX® (QIAGEN Group). 1066980 01/2011 © 2011 QIAGEN, all rights reserved.

4. Incubate at 50°C for 10 min to solubilize the agarose and bind the DNA. Mix by vortexing* every 2 min to keep QIAEX II in suspension. Check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color should turn to yellow. The incubation should then be continued for at least 5 min.

5. Centrifuge the sample for 30 s and carefully remove supernatant with a pipet.

6. Wash the pellet with 500 μl Buffer QX1. Resuspend the pellet by vortexing.* Centrifuge the sample for 30 s and remove all traces of supernatant with a pipet. This wash step removes residual agarose contaminants.

7. Wash the pellet twice with 500 μl Buffer PE. Resuspend the pellet by vortexing.* Centrifuge the sample for 30 s and carefully remove all traces of supernatant with a pipet. This step removes residual salt contaminants.

8. Air-dry the pellet for 10–15 min or until the pellet becomes white. If 30 μl QIAEX II suspension is used, air-dry the pellet for approximately 30 min. Do not vacuum dry, as overdrying, may lead to decreased elution efficiency.

9. To elute DNA, add 20 μl of 10 mM Tris·Cl, pH 8.5, TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), or water and resuspend the pellet by vortexing.* Incubate according to the DNA fragment size: 5 min at room temperature (15–25°C) for ≤4 kb; 5 min at 50°C for 4–10 kb; or 10 min at 50°C for >10 kb.

10. Centrifuge for 30 s, and carefully pipet the supernatant into a clean tube. The supernatant now contains the purified DNA.

11. Optional: repeat steps 9 and 10 and combine the eluates. A second elution step will increase the yield by approximately 10–15%.