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 scapel

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 thet 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 twice with 500µl of Buffer PE

Air-dsy 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 fpr 30 s. Carefully pipet the supernatant into a clean tube

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