Difference between revisions of "Fragment Purification"

Revision as of 15:02, 1 February 2014

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 600 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 1 minute at full speed
7. Dump the collection tube, transfer the remaining volume to the column, and spin 1 minute
8. Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) 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 55 C water bath
15. Dump the collection tube and spin the column at full speed for 1 minute
16. Transfer the dry column to a new 1.5 ml tube
17. Add 15 ul (or less if higher concentration is desired, eg. 12 ul or even 8 ul) heated NE elution buffer to the column and let stand 1 minute
18. Spin full speed for 30 seconds
19. Reapply the eluate onto the column and let stand 1 minute
20. Spin full speed 60 seconds
21. Measure concentration on Nanodrop or Cytation 3