Fragment Purification
From GcatWiki
Fragment Purification from an Agarose Gel
Nucleospin Extract IIkit from Machery-Nagel
- Add 5X loading buffer to the restriction digest (eg. 40 ul digest + 10 ul 5X) and run on 1% Agarose gel
- 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
- Optional: Weigh the gel slice (expect 100-250 mg)and add 2 volumes of buffer NTI
- Easier to just add 700 ul of buffer NTI
- Incubate 5 minutes at 55 C, vortexing 2 times along the way and at the end
- Transfer into a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 1 minute at full speed
- Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column
- Spin 30 sec at full speed
- Repeat steps 8 and 9
- Dump collection tube and add 250 ul wash buffer NT3 to column
- Spin 30 sec at full speed
- Repeat steps 11 and 12
- Place a small tube of NE elution buffer in the 55 C water bath
- Dump the collection tube and spin the column at full speed for 1 minute
- At 15 ul heated NE elution buffer to the dry column and let stand 1 minute
- Spin full speed for 30 seconds
- Reapply the eluate onto the column and let stand 1 minute
- Spin full speed 30 seconds
- Measure concentration on Nanodrop or Cytation 3