Fragment Purification - Revision history

Eckdahl at 14:13, 19 May 2015

2015-05-19T14:13:42Z

Eckdahl at 15:02, 1 February 2014

2014-02-01T15:02:38Z

Eckdahl at 14:42, 1 February 2014

2014-02-01T14:42:59Z

Eckdahl at 14:36, 1 February 2014

2014-02-01T14:36:04Z

Eckdahl at 14:34, 1 February 2014

2014-02-01T14:34:39Z

Eckdahl at 17:46, 7 June 2010

2010-06-07T17:46:58Z

Eckdahl at 17:43, 7 June 2010

2010-06-07T17:43:09Z

New page

'''Fragment Purification from an 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
#Weigh the gel slice (expect 100-250 mg)
#Use the [http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel
#Add 2 volumes of buffer NT
#Incubate 5 minutes at 50 C, vortexing 2 times along the way and at the end
#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 buffer NT3 to column
#Spin 30 sec at full speed

@@ Line 3: / Line 3: @@
 [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

@@ Line 19: / Line 19: @@
 #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
+#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 30 seconds
+#Spin full speed 60 seconds
 #Measure concentration on Nanodrop or Cytation 3

@@ Line 8: / Line 8: @@
 #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
+#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
 #Spin 30 sec at full speed

@@ Line 17: / Line 17: @@
 #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
+#Transfer the dry column to a new 1.5 ml tube
 #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

@@ Line 1: / Line 1: @@
 '''Fragment Purification from an 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
-#Use the [http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel
+#Easier to just add 700 ul of buffer NTI
-#Add 2 volumes of buffer NT
+#Incubate 5 minutes at 55 C, vortexing 2 times along the way and at the end
-#Incubate 5 minutes at 50 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
-#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
 #Spin 30 sec at full speed
 #Repeat steps 8 and 9
@@ Line 14: / Line 15: @@
 #Spin 30 sec at full speed
 #Repeat steps 11 and 12
-#Place a small tube of NE elution buffer in the 65 C water bath
+#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
@@ Line 20: / Line 21: @@
 #Reapply the eluate onto the column and let stand 1 minute
 #Spin full speed 30 seconds
-#Measure concentration on Nanodrop
+#Measure concentration on Nanodrop or Cytation 3