Muratore:Protocols/PCR/Quikchange - Revision history

Kathryn Muratore at 21:05, 23 June 2011

Kathryn Muratore — Thu, 23 Jun 2011 21:05:42 GMT

←Older revision		Revision as of 21:05, 23 June 2011
Line 25:		Line 25:

	==2-stage QuikChange protocol==		==2-stage QuikChange protocol==
-	This protocol is based on the report by Wang & Malcolm. (1999) ~~Biotechniques '''26''':680-682~~. It is especially helpful for large inserts.	+	This protocol is based on the report by {{Ref\|Wang & Malcolm (1999)}}. It is especially helpful for large inserts.
	{\|border="1"		{\|border="1"
	\|+		\|+
Line 76:		Line 76:
	:# 15' @ 68°C		:# 15' @ 68°C
	:# hold @ 4°C		:# hold @ 4°C
		+
		+	===References===
		+	# {{FormatRef\|Wang & Malcolm\|1999\| \|Biotechniques 26\|680\| }}

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:DNA]]		[[Category:DNA]]
	[[Category:PCR]]		[[Category:PCR]]

Kathryn Muratore: Muratore:Protocols/PCR/QuikChange moved to Muratore:Protocols/PCR/Quikchange: Conforming to naming standards

Kathryn Muratore — Thu, 23 Jun 2011 17:41:49 GMT

Muratore:Protocols/PCR/QuikChange moved to Muratore:Protocols/PCR/Quikchange: Conforming to naming standards

←Older revision

Revision as of 17:41, 23 June 2011

Kathryn Muratore at 16:04, 9 June 2011

Kathryn Muratore — Thu, 09 Jun 2011 16:04:45 GMT

←Older revision		Revision as of 16:04, 9 June 2011
Line 1:		Line 1:
		+	==Basic QuikChange protocol==
	{\|border="1"		{\|border="1"
	\|+		\|+
Line 8:		Line 9:
	\|-		\|-
	!Experimental		!Experimental
-	\|12 μL \|\|5 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|1 μL \|\|50 μL	+	\|17 μL \|\|5 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|1 μL \|\|50 μL
	\|-		\|-
	!(-) Control		!(-) Control
-	\|13 μL \|\|5 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|0 μL \|\|50 μL	+	\|18 μL \|\|5 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|0 μL \|\|50 μL
	\|}		\|}
	:* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.		:* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
Line 20:		Line 21:
	:# 1'/kb @ 68°C		:# 1'/kb @ 68°C
	:# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times		:# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
		+	:# 15' @ 68°C
		+	:# hold @ 4°C
		+
		+	==2-stage QuikChange protocol==
		+	This protocol is based on the report by Wang & Malcolm. (1999) Biotechniques '''26''':680-682. It is especially helpful for large inserts.
		+	{\|border="1"
		+	\|+
		+	'''1st stage'''
		+	\|-
		+	!Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!For primer !!Rev primer !!dNTPs !!Pfu Turbo !!wax
		+	\|-
		+	! !! !!(10X) !!(10 μg/mL) !!(12.5 ng/μL) !!(12.5 ng/μL) !!(10 mM ea) !!(2.5 U/μL) !!
		+	\|-
		+	!Experimental-forward
		+	\|27 μL \|\|5 μL \|\|5 μL \|\|10 μL \|\|0 μL \|\|2 μL \|\|1 μL \|\|50 μL
		+	\|-
		+	!Experimental-reverse
		+	\|27 μL \|\|5 μL \|\|5 μL \|\|0 μL \|\|10 μL \|\|2 μL \|\|1 μL \|\|50 μL
		+	\|-
		+	!(-) Control-forward
		+	\|28 μL \|\|5 μL \|\|5 μL \|\|10 μL \|\|0 μL \|\|2 μL \|\|0 μL \|\|50 μL
		+	\|-
		+	!(-) Control-reverse
		+	\|28 μL \|\|5 μL \|\|5 μL \|\|0 μL \|\|10 μL \|\|2 μL \|\|0 μL \|\|50 μL
		+	\|}
		+	:* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
		+	:* dNTPs should be aliquoted to minimize freeze-thaw cycles and ensure the integrity of the triphosphate.
		+	:# 30" @ 95°C
		+	:# 30" @ 95°C
		+	:# 1' @ 55°C
		+	:# 1'/kb @ 68°C
		+	:# remove from PCR machine, pipet off wax, and chill briefly to re-harden thin wax layer
		+	:# go immediately to 2nd stage:
		+	{\|border="1"
		+	\|+
		+	'''2nd stage'''
		+	\|-
		+	!Tube !!1st stage-forward !!1st stage-reverse !!Pfu Turbo !!wax
		+	\|-
		+	! !! !! !!(2.5 U/μL) !!
		+	\|-
		+	!Experimental
		+	\|25 μL \|\|25 μL \|\|1 μL \|\|50 μL
		+	\|-
		+	!(-) Control
		+	\|25 μL \|\|25 μL \|\|0 μL \|\|50 μL
		+	\|}
		+	:* Mix DNA and Pfu by pipetting, then add wax.
		+	:# 30" @ 95°C
		+	:# 30" @ 95°C
		+	:# 1' @ 55°C
		+	:# 1'/kb @ 68°C
		+	:# repeat steps 2-4 x 17 more times
	:# 15' @ 68°C		:# 15' @ 68°C
	:# hold @ 4°C		:# hold @ 4°C

Kathryn Muratore: wrong volumes in table and added note about dNTPs

Kathryn Muratore — Thu, 09 Jun 2011 15:33:41 GMT

wrong volumes in table and added note about dNTPs

←Older revision		Revision as of 15:33, 9 June 2011
Line 8:		Line 8:
	\|-		\|-
	!Experimental		!Experimental
-	\|12 μL \|\|10 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|1 μL \|\|50 μL	+	\|12 μL \|\|5 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|1 μL \|\|50 μL
	\|-		\|-
	!(-) Control		!(-) Control
-	\|13 μL \|\|10 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|0 μL \|\|50 μL	+	\|13 μL \|\|5 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|0 μL \|\|50 μL
	\|}		\|}
	:* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.		:* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
		+	:* dNTPs should be aliquoted to minimize freeze-thaw cycles and ensure the integrity of the triphosphate.
	:# 30" @ 95°C		:# 30" @ 95°C
	:# 30" @ 95°C		:# 30" @ 95°C

Kathryn Muratore at 02:21, 8 June 2011

Kathryn Muratore — Wed, 08 Jun 2011 02:21:05 GMT

←Older revision		Revision as of 02:21, 8 June 2011
Line 8:		Line 8:
	\|-		\|-
	!Experimental		!Experimental
-	\|12 μL \|\|10 μL \|\|10 μL \|\|10 μL \|\|5 μL \|\|2 μL \|\|1 μL \|\|50 μL	+	\|12 μL \|\|10 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|1 μL \|\|50 μL
	\|-		\|-
	!(-) Control		!(-) Control
-	\|13 μL \|\|10 μL \|\|10 μL \|\|10 μL \|\|5 μL \|\|2 μL \|\|0 μL \|\|50 μL	+	\|13 μL \|\|10 μL \|\|5 μL \|\|10 μL \|\|10 μL \|\|2 μL \|\|0 μL \|\|50 μL
	\|}		\|}
	:* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.		:* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
Line 17:		Line 17:
	:# 30" @ 95°C		:# 30" @ 95°C
	:# 1' @ 55°C		:# 1' @ 55°C
-	:# ~~8.5~~' @ 68°C	+	:# 1'/kb @ 68°C
	:# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times		:# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
-	:# 10' @ 68°C	+	:# 15' @ 68°C
		+	:# hold @ 4°C

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:DNA]]		[[Category:DNA]]
	[[Category:PCR]]		[[Category:PCR]]

Kathryn Muratore at 18:03, 26 May 2011

Kathryn Muratore — Thu, 26 May 2011 18:03:05 GMT

←Older revision		Revision as of 18:03, 26 May 2011
Line 13:		Line 13:
	\|13 μL \|\|10 μL \|\|10 μL \|\|10 μL \|\|5 μL \|\|2 μL \|\|0 μL \|\|50 μL		\|13 μL \|\|10 μL \|\|10 μL \|\|10 μL \|\|5 μL \|\|2 μL \|\|0 μL \|\|50 μL
	\|}		\|}
-	* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.	+	:* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
-	# 30" @ 95°C	+	:# 30" @ 95°C
-	# 30" @ 95°C	+	:# 30" @ 95°C
-	# 1' @ 55°C	+	:# 1' @ 55°C
-	# 8.5' @ 68°C	+	:# 8.5' @ 68°C
-	# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times	+	:# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
-	# 10' @ 68°C	+	:# 10' @ 68°C

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:DNA]]		[[Category:DNA]]
	[[Category:PCR]]		[[Category:PCR]]

Kathryn Muratore: New page: {|border="1" |+ '''Basic QuikChange protocol''' |- !Tube !!sterile H₂O !!Pfu Buffer !!Template !!For primer !!Rev primer !!dNTPs !!Pfu Turbo !!wax |- ! !! !!(10X) !!(10 μg/mL) ...

Kathryn Muratore — Thu, 26 May 2011 18:01:24 GMT

New page: {|border="1" |+ '''Basic QuikChange protocol''' |- !Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!For primer !!Rev primer !!dNTPs !!Pfu Turbo !!wax |- ! !! !!(10X) !!(10 μg/mL) ...

New page

{|border="1"
|+
'''Basic QuikChange protocol'''
|-
!Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!For primer !!Rev primer !!dNTPs !!Pfu Turbo !!wax
|-
! !! !!(10X) !!(10 μg/mL) !!(12.5 ng/μL) !!(12.5 ng/μL) !!(10 mM ea) !!(2.5 U/μL) !!
|-
!Experimental
|12 μL ||10 μL ||10 μL ||10 μL ||5 μL ||2 μL ||1 μL ||50 μL
|-
!(-) Control
|13 μL ||10 μL ||10 μL ||10 μL ||5 μL ||2 μL ||0 μL ||50 μL
|}
* Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
# 30" @ 95°C
# 30" @ 95°C
# 1' @ 55°C
# 8.5' @ 68°C
# repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
# 10' @ 68°C

[[Category:Protocol]]
[[Category:DNA]]
[[Category:PCR]]