Muratore:Protocols/PCR/Quikchange

Basic QuikChange protocol

**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	17 μL	5 μL	5 μL	10 μL	10 μL	2 μL	1 μL	50 μL
(-) Control	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.
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
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

This protocol is based on the report by Wang & Malcolm (1999). It is especially helpful for large inserts.

**1st stage**
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-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:

**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