Fragment Purification

Fragment Purification from an Agarose Gel

Nucleospin Extract IIkit from Machery-Nagel

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 and place in labeled 1.5 ml tube
3. Optional: Weigh the gel slice (expect 100-250 mg)and add 2 volumes of buffer NTI
4. Easier to just add 700 ul of buffer NTI
5. Incubate 5 minutes at 55 C, vortexing 2 times along the way and at the end
6. Transfer into a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 1 minute at full speed
7. Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column
8. Spin 30 sec at full speed
9. Repeat steps 8 and 9
10. Dump collection tube and add 250 ul wash buffer NT3 to column
11. Spin 30 sec at full speed
12. Repeat steps 11 and 12
13. Place a small tube of NE elution buffer in the 55 C water bath
14. Dump the collection tube and spin the column at full speed for 1 minute
15. At 15 ul heated NE elution buffer to the dry column and let stand 1 minute
16. Spin full speed for 30 seconds
17. Reapply the eluate onto the column and let stand 1 minute
18. Spin full speed 30 seconds
19. Measure concentration on Nanodrop or Cytation 3