Difference between revisions of "Fragment Purification"
From GcatWiki
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'''Fragment Purification from an Agarose Gel''' | '''Fragment Purification from an Agarose Gel''' | ||
| + | |||
| + | [http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel | ||
#Add 5X loading buffer to the restriction digest (eg. 40 ul digest + 10 ul 5X) and run on 1% Agarose gel | #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 | + | #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 |
| − | #Weigh the gel slice (expect 100-250 mg) | + | #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 | |
| − | #Incubate 5 minutes at | + | #Transfer into a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 1 minute at full speed |
| − | #Transfer into a Nucleospin Extract II 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 |
| − | #Dump collection tube and add 700 ul wash buffer NT3 to column | ||
#Spin 30 sec at full speed | #Spin 30 sec at full speed | ||
#Repeat steps 8 and 9 | #Repeat steps 8 and 9 | ||
| Line 14: | Line 15: | ||
#Spin 30 sec at full speed | #Spin 30 sec at full speed | ||
#Repeat steps 11 and 12 | #Repeat steps 11 and 12 | ||
| − | #Place a small tube of NE elution buffer in the | + | #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 | #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 | #At 15 ul heated NE elution buffer to the dry column and let stand 1 minute | ||
| Line 20: | Line 21: | ||
#Reapply the eluate onto the column and let stand 1 minute | #Reapply the eluate onto the column and let stand 1 minute | ||
#Spin full speed 30 seconds | #Spin full speed 30 seconds | ||
| − | #Measure concentration on Nanodrop | + | #Measure concentration on Nanodrop or Cytation 3 |
Revision as of 14:34, 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 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
