Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction

gel extraction with the QIAEX II Gel Extraction Kit (150)

source: http://www.qiagen.com/literature/render.aspx?id=130

1. Excise the DNA band from the agarose gel with a clean, sharp scalpel

2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QX1 to 1 volume of gel (for DNA fragments 100bp - 4kb) (example: 100mg gel -> 300 µl of Buffer QX1)

3. Resuspend QIAEX II by vortexing for 30 s. Add QIAEX II to the sample according to the table below and mix

<= 2 µg DNA	Add 10 µl of QIAEX II
2 - 10 µg DNA	Add 30 µl of QIAEX II
Each additional 10 µg DNA	Add additional 30 µl of QIAEX II

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

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

6. Wash the pellet with 500 µl of Buffer QX1 (resuspend pellet by vortexing)

7. Wash the pellet twice with 500 µl of Buffer PE (resuspend pellet by vortexing)

8. Air-dry the pellet for 10 - 15 min or until the pellet becomes white

9. To elute DNA, add 20 µl of 10 mM Tris-Cl, pH 8.5 of H₂O and resuspend the pellet by vortexing. Incubate according to the following table:

DNA fragments <= 4 kb	Incubate at room temp. for 5 min
DNA fragments 4 - 10 kb	Incubate at 50°C for 5 min
DNA fragments > 10 kb	Incubate at 59°C for 10 min

10. Centrifuge for 30 s. Carefully pipet the supernatant into a clean tube

11. Optional: repeat steps 9 and 10 and combine the eluates