Difference between revisions of "Fragment Purification"
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#Easier to just add 700 ul 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 | #Incubate 5 minutes at 55 C, vortexing 2 times along the way and at the end | ||
− | #Transfer | + | #Transfer 600 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 1 minute at full speed |
+ | #Dump the collection tube, transfer the remaining volume to the column, and spin 1 minute | ||
#Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column | #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 | #Spin 30 sec at full speed |
Revision as of 14:42, 1 February 2014
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 600 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 1 minute at full speed
- Dump the collection tube, transfer the remaining volume to the column, and spin 1 minute
- 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
- Transfer the dry column to a new 1.5 ml tube
- Add 15 ul heated NE elution buffer to the 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