Knight:PhoenIX Maxiprep Kit - Revision history

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Thu, 11 Jan 2007 01:06:17 GMT

Procedure

←Older revision		Revision as of 01:06, 11 January 2007
Line 20:		Line 20:
	#*''Trace media can affect subsequent steps.''		#*''Trace media can affect subsequent steps.''
	#Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended.		#Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended.
-	#*There is some resuspension buffer with RNase A added stored at 4°C in 32-306. To make more, you'll need to add RNase A to more resuspension buffer.	+	#*There is some resuspension buffer with RNase A added stored at 4°C in 32-306. To make more, you'll need to add RNase A to more resuspension buffer. The resuspension buffer (without RNase A) is in the box and the RNase A is stored at -20°C on the door.
	#Transfer resuspended cells to 50 mL conical tube.		#Transfer resuspended cells to 50 mL conical tube.
	#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.		#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Thu, 11 Jan 2007 01:04:42 GMT

Procedure

←Older revision		Revision as of 01:04, 11 January 2007
Line 20:		Line 20:
	#*''Trace media can affect subsequent steps.''		#*''Trace media can affect subsequent steps.''
	#Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended.		#Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended.
		+	#*There is some resuspension buffer with RNase A added stored at 4°C in 32-306. To make more, you'll need to add RNase A to more resuspension buffer.
	#Transfer resuspended cells to 50 mL conical tube.		#Transfer resuspended cells to 50 mL conical tube.
	#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.		#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.

Reshma P. Shetty at 20:59, 10 December 2006

Reshma P. Shetty — Sun, 10 Dec 2006 20:59:55 GMT

←Older revision		Revision as of 20:59, 10 December 2006
Line 57:		Line 57:
	*These spin steps may not be hard enough. Most of the purification steps are supposed to be at 12,000 x g.		*These spin steps may not be hard enough. Most of the purification steps are supposed to be at 12,000 x g.
	*You're not supposed to let the column stand between steps.		*You're not supposed to let the column stand between steps.
		+
		+	[[Category:Protocol]]
		+	[[Category:In vitro]]
		+	[[Category:DNA]]

Reshma P. Shetty: /* Materials */

Reshma P. Shetty — Sun, 10 Dec 2006 20:58:29 GMT

Materials

←Older revision		Revision as of 20:58, 10 December 2006
Line 3:		Line 3:
	*Isopropanol		*Isopropanol
	*70% Ethanol		*70% Ethanol
-	*[[5810R centrifuge]]	+	*[[Knight:5810R centrifuge]]

	==When using a new kit==		==When using a new kit==

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Sun, 10 Dec 2006 20:57:37 GMT

Procedure

Reshma P. Shetty at 20:54, 10 December 2006

Reshma P. Shetty — Sun, 10 Dec 2006 20:54:46 GMT

←Older revision		Revision as of 20:54, 10 December 2006
Line 1:		Line 1:
-
	==Materials==		==Materials==
	*[http://www.qbiogene.com/products/phoenix/ph_maxiprep.shtml PhoenIX™ Maxiprep Kit]		*[http://www.qbiogene.com/products/phoenix/ph_maxiprep.shtml PhoenIX™ Maxiprep Kit]
	*Isopropanol		*Isopropanol
-	#70% Ethanol	+	*70% Ethanol
		+	*[[5810R centrifuge]]
		+
		+	==When using a new kit==
		+	#Assemble cardboard column rack.
		+	#Note that the resuspension, lysis and neutralization buffer bottles are VERY hard to open.

	==Procedure==		==Procedure==
-	See [http://www.qbiogene.com/technical/protocols/Phoenix/PhoenIX%20maxi%20kit.pdf ~~normal~~ protocol] and ~~[http~~:~~//www~~.~~qbiogene~~.~~com/technical/protocols/Phoenix/PhoenIX~~%~~20MaxiLowRefill~~.~~pdf low copy protocol]~~.	+	See [http://www.qbiogene.com/technical/protocols/Phoenix/PhoenIX%20maxi%20kit.pdf official protocol]. The steps below have been changed to accommodate our tubes and centrifuge.
		+
		+	#Place PhoenIX™ Maxi column in the assembled column rack.
		+	#Place a beaker underneath to collect flow through.
		+	#Add 30 ml of equilibration buffer (gray cap label) to the surface of the column and allow the liquid to
		+	drain by gravity flow.
		+	#*''Note: It will take 15-20 minutes for the column to drain completely.''
		+	#Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4°C.
		+	#Remove all traces of liquid medium from the bacterial cell pellet by pouring.
		+	#*''Trace media can affect subsequent steps.''
		+	#Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and
		+	vortex until completely resuspended.
		+	#Transfer resuspended cells to 50 mL conical tube.
		+	#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.
		+	#Mix thoroughly by inverting until the lysate appears to be homogeneous (5-6 inversions). DO NOT VORTEX.
		+	#Incubate 5 minutes at room temperature.
		+	#*''Note: Do not incubate for longer than 5 minutes or plasmid DNA might become irreversibly denatured.''
		+	#Add 10 ml of neutralization buffer (green cap label).
		+	#Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained. DO NOT VORTEX.
		+	#*''Note: If preparing several samples at once, thoroughly mix each sample immediately after the addition of
		+	the neutralization buffer before adding the buffer to the next tube.''
		+	#Centrifuge at 9,000 x g for 20 mins at room temperature.
		+	#''Note: The supernatant must at room temperature (18 – 25°C) prior to loading on the column.''
		+	#Verify that the qquilibration buffer has been collected in the beaker.
		+	#Discard the flow-through and replace the container.
		+	#Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column.
		+	#*''Note: Do not pour lysate directly onto the column. Use a pipette to ensure that precipitate particles do not
		+	enter the column and cause clogging.''
		+	#Allow the lysate to drain by gravity flow (10-15 minutes).
		+	#Discard the flowthrough and replace the empty container.
		+	#Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to
		+	drain by gravity flow (20-25 minutes).
		+	#Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to
		+	drain by gravity flow (20-25 minutes).
		+	#Discard the flow-through.
		+	#Replace the waste collection container with a 50 mL conical tube.
		+	#Add 15 ml of elution buffer (pink cap label) to the top of the column.
		+	#Allow the eluate to drain by gravity flow (5-10 minutes) into the centrifuge tube.
		+	#Add 10.5 ml of room temperature isopropanol to the eluted plasmid DNA in the centrifuge tube.
		+	#Mix and centrifuge at 9,000 x g for 40 minutes at 4°C.
		+	#Pour out the supernatant taking care not to disturb the DNA pellet.
		+	#Add 5 ml of room temperature 70% ethanol and wash the pellet.
		+	#Centrifuge at 9,000 x g for 10 minutes at 4°C.
		+	#Completely remove ALL of the supernatant from the pellet with a pipette.
		+	#Air-dry the pellet for 10 minutes.
		+	#''Note: Drying with a vacuum chamber is not recommended because over-dried DNA may be difficult to completely resuspend.''
		+	#Dissolve the plasmid DNA in 500 μl of water.
		+	#Move to smaller tube.
		+	#Take a spectrophotometer reading to assess concentration.

Reshma P. Shetty at 20:20, 10 December 2006

Reshma P. Shetty — Sun, 10 Dec 2006 20:20:12 GMT

New page

==Materials==
*[http://www.qbiogene.com/products/phoenix/ph_maxiprep.shtml PhoenIX™ Maxiprep Kit]
*Isopropanol
#70% Ethanol

==Procedure==
See [http://www.qbiogene.com/technical/protocols/Phoenix/PhoenIX%20maxi%20kit.pdf normal protocol] and [http://www.qbiogene.com/technical/protocols/Phoenix/PhoenIX%20MaxiLowRefill.pdf low copy protocol].

←Older revision		Revision as of 20:57, 10 December 2006
Line 14:		Line 14:
	#Place PhoenIX™ Maxi column in the assembled column rack.		#Place PhoenIX™ Maxi column in the assembled column rack.
	#Place a beaker underneath to collect flow through.		#Place a beaker underneath to collect flow through.
-	#Add 30 ml of equilibration buffer (gray cap label) to the surface of the column and allow the liquid to	+	#Add 30 ml of equilibration buffer (gray cap label) to the surface of the column and allow the liquid to drain by gravity flow.
-	drain by gravity flow.	+
	#*''Note: It will take 15-20 minutes for the column to drain completely.''		#*''Note: It will take 15-20 minutes for the column to drain completely.''
	#Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4°C.		#Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4°C.
	#Remove all traces of liquid medium from the bacterial cell pellet by pouring.		#Remove all traces of liquid medium from the bacterial cell pellet by pouring.
	#*''Trace media can affect subsequent steps.''		#*''Trace media can affect subsequent steps.''
-	#Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and	+	#Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended.
-	vortex until completely resuspended.	+
	#Transfer resuspended cells to 50 mL conical tube.		#Transfer resuspended cells to 50 mL conical tube.
	#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.		#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.
Line 29:		Line 27:
	#Add 10 ml of neutralization buffer (green cap label).		#Add 10 ml of neutralization buffer (green cap label).
	#Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained. DO NOT VORTEX.		#Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained. DO NOT VORTEX.
-	#*''Note: If preparing several samples at once, thoroughly mix each sample immediately after the addition of	+	#*''Note: If preparing several samples at once, thoroughly mix each sample immediately after the addition of the neutralization buffer before adding the buffer to the next tube.''
-	the neutralization buffer before adding the buffer to the next tube.''	+
	#Centrifuge at 9,000 x g for 20 mins at room temperature.		#Centrifuge at 9,000 x g for 20 mins at room temperature.
-	#''Note: The supernatant must at room temperature (18 – 25°C) prior to loading on the column.''	+	#*''Note: The supernatant must at room temperature (18 – 25°C) prior to loading on the column.''
	#Verify that the qquilibration buffer has been collected in the beaker.		#Verify that the qquilibration buffer has been collected in the beaker.
	#Discard the flow-through and replace the container.		#Discard the flow-through and replace the container.
	#Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column.		#Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column.
-	#*''Note: Do not pour lysate directly onto the column. Use a pipette to ensure that precipitate particles do not	+	#*''Note: Do not pour lysate directly onto the column. Use a pipette to ensure that precipitate particles do not enter the column and cause clogging.''
-	enter the column and cause clogging.''	+
	#Allow the lysate to drain by gravity flow (10-15 minutes).		#Allow the lysate to drain by gravity flow (10-15 minutes).
	#Discard the flowthrough and replace the empty container.		#Discard the flowthrough and replace the empty container.
-	#Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to	+	#Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to drain by gravity flow (10 minutes).
-	drain by gravity flow (~~20-25~~ minutes).	+	#Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to drain by gravity flow (10 minutes).
-	#Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to	+
-	drain by gravity flow (~~20-25~~ minutes).	+
	#Discard the flow-through.		#Discard the flow-through.
	#Replace the waste collection container with a 50 mL conical tube.		#Replace the waste collection container with a 50 mL conical tube.
Line 55:		Line 49:
	#Completely remove ALL of the supernatant from the pellet with a pipette.		#Completely remove ALL of the supernatant from the pellet with a pipette.
	#Air-dry the pellet for 10 minutes.		#Air-dry the pellet for 10 minutes.
-	#''Note: Drying with a vacuum chamber is not recommended because over-dried DNA may be difficult to completely resuspend.''	+	#*''Note: Drying with a vacuum chamber is not recommended because over-dried DNA may be difficult to completely resuspend.''
	#Dissolve the plasmid DNA in 500 μl of water.		#Dissolve the plasmid DNA in 500 μl of water.
	#Move to smaller tube.		#Move to smaller tube.
	#Take a spectrophotometer reading to assess concentration.		#Take a spectrophotometer reading to assess concentration.
		+
		+	==Notes==
		+	*These spin steps may not be hard enough. Most of the purification steps are supposed to be at 12,000 x g.
		+	*You're not supposed to let the column stand between steps.