Difference between revisions of "Fragment Purification"

Revision as of 17:46, 7 June 2010

Fragment Purification from an Agarose Gel

1. Add 5X loading buffer to the restriction digest (eg. 40 ul digest + 10 ul 5X) and run on 1% Agarose gel
2. With a plastic ruler, and wearing safety glasses, slice the band out of the gel on the UV box
3. Weigh the gel slice (expect 100-250 mg)
4. Use the Nucleospin Extract IIkit from Machery-Nagel
5. Add 2 volumes of buffer NT
6. Incubate 5 minutes at 50 C, vortexing 2 times along the way and at the end
7. Transfer into a Nucleospin Extract II column on a collection tube and spin 1 minute at full speed
8. Dump collection tube and add 700 ul wash buffer NT3 to column
9. Spin 30 sec at full speed
10. Repeat steps 8 and 9
11. Dump collection tube and add 250 ul wash buffer NT3 to column
12. Spin 30 sec at full speed
13. Repeat steps 11 and 12
14. Place a small tube of NE elution buffer in the 65 C water bath
15. Dump the collection tube and spin the column at full speed for 1 minute
16. At 15 ul heated NE elution buffer to the dry column and let stand 1 minute
17. Spin full speed for 30 seconds
18. Reapply the eluate onto the column and let stand 1 minute
19. Spin full speed 30 seconds
20. Measure concentration on Nanodrop