Muratore:Protocols/PCR/Quikchange: Difference between revisions

Revision as of 11:03, 26 May 2011

**Basic QuikChange protocol**
Tube	sterile H₂O	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

@@ Line 13: / Line 13: @@
 |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:DNA]]
 [[Category:PCR]]