Difference between revisions of "Fragment Purification"

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[http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel
 
[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
+
#With a plastic ruler cut band out of agarose gel and place in 1.5 microcentrifuge tube.
#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
+
#Add 700 ul of buffer NTI and incubate at least 5 minutes in 55 C water bath, vortexing every minute until gel slice disappears.
#Optional: Weigh the gel slice (expect 100-250 mg)and add 2 volumes of buffer NTI
+
#Incubate a dH20 in 55 C water bath for at later use.
#Easier to just add 700 ul of buffer NTI
+
#Transfer 700 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 30 seconds at full speed  
#Incubate 5 minutes at 55 C, vortexing 2 times along the way and at the end
+
#Dump the collection tube, transfer the remaining volume to the column, and spin 30 seconds.
#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 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.
#Dump the collection tube, transfer the remaining volume to the column, and spin 1 minute
+
#Repeat step 6.
#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 250 ul wash buffer NT3 to column, then spin 30 seconds.
#Spin 30 sec at full speed
+
#Repeat step 8.
#Repeat steps 8 and 9
+
#Dump the collection tube and spin the column at full speed for 1 minute to dry colum.
#Dump collection tube and add 250 ul wash buffer NT3 to column
+
#Transfer to a NEW 1.5 ml tube.
#Spin 30 sec at full speed
+
#Add 12 ul heated dH2O directly to filter, let stand 30 seconds, and spin 30 seconds.
#Repeat steps 11 and 12
+
#Reapply the eluate onto the column, let stand 1 minute, spin 1 minute.
#Place a small tube of NE elution buffer in the 55 C water bath
+
#Measure concentration on Nanodrop
#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 (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
 
#Spin full speed for 30 seconds
 
#Reapply the eluate onto the column and let stand 1 minute
 
#Spin full speed 60 seconds
 
#Measure concentration on Nanodrop or Cytation 3
 

Latest revision as of 12:13, 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