Difference between revisions of "Fragment Purification"

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'''Fragment Purification from an Agarose Gel'''
 
'''Fragment Purification from an Agarose Gel'''
  
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#Add 5X loading buffer to the restriction digest (eg. 40 ul digest + 10 ul 5X) and run on 1% Agarose gel
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[http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel
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#With a plastic ruler, and wearing safety glasses, slice the band out of the gel on the UV box
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#Weigh the gel slice (expect 100-250 mg)
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#With a plastic ruler cut band out of agarose gel and place in 1.5 microcentrifuge tube.
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#Use the [http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel
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#Add 700 ul of buffer NTI and incubate at least 5 minutes in 55 C water bath, vortexing every minute until gel slice disappears.
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#Add 2 volumes of buffer NT
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#Incubate a dH20 in 55 C water bath for at later use.
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#Incubate 5 minutes at 50 C, vortexing 2 times along the way and at the end
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#Transfer 700 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 30 seconds at full speed  
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#Transfer into a Nucleospin Extract II column on a collection tube and spin 1 minute at full speed
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#Dump the collection tube, transfer the remaining volume to the column, and spin 30 seconds.
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#Dump collection tube and add 700 ul wash buffer NT3 to column
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#Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column, then spin 30 seconds.
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#Spin 30 sec at full speed
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#Repeat step 6.
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#Repeat steps 8 and 9
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#Dump collection tube and add 250 ul wash buffer NT3 to column, then spin 30 seconds.
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#Dump collection tube and add 250 ul wash buffer NT3 to column
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#Repeat step 8.
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#Spin 30 sec at full speed
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#Dump the collection tube and spin the column at full speed for 1 minute to dry colum.
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#Repeat steps 11 and 12
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#Transfer to a NEW 1.5 ml tube.
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#Place a small tube of NE elution buffer in the 65 C water bath
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#Add 12 ul heated dH2O directly to filter, let stand 30 seconds, and spin 30 seconds.
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#Dump the collection tube and spin the column at full speed for 1 minute
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#Reapply the eluate onto the column, let stand 1 minute, spin 1 minute.
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#At 15 ul heated NE elution buffer to the dry column and let stand 1 minute
 
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#Spin full speed for 30 seconds
 
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#Reapply the eluate onto the column and let stand 1 minute
 
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#Spin full speed 30 seconds
 
 
#Measure concentration on Nanodrop
 
#Measure concentration on Nanodrop

Latest revision as of 14:13, 19 May 2015

Fragment Purification from an Agarose Gel

Nucleospin Extract IIkit from Machery-Nagel

  1. With a plastic ruler cut band out of agarose gel and place in 1.5 microcentrifuge tube.
  2. Add 700 ul of buffer NTI and incubate at least 5 minutes in 55 C water bath, vortexing every minute until gel slice disappears.
  3. Incubate a dH20 in 55 C water bath for at later use.
  4. Transfer 700 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 30 seconds at full speed
  5. Dump the collection tube, transfer the remaining volume to the column, and spin 30 seconds.
  6. Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column, then spin 30 seconds.
  7. Repeat step 6.
  8. Dump collection tube and add 250 ul wash buffer NT3 to column, then spin 30 seconds.
  9. Repeat step 8.
  10. Dump the collection tube and spin the column at full speed for 1 minute to dry colum.
  11. Transfer to a NEW 1.5 ml tube.
  12. Add 12 ul heated dH2O directly to filter, let stand 30 seconds, and spin 30 seconds.
  13. Reapply the eluate onto the column, let stand 1 minute, spin 1 minute.
  14. Measure concentration on Nanodrop
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