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.

1. 30" @ 95°C
2. 30" @ 95°C
3. 1' @ 55°C
4. 1'/kb @ 68°C
5. repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
6. 15' @ 68°C
7. 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.

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.

1. 30" @ 95°C
2. 30" @ 95°C
3. 1' @ 55°C
4. 1'/kb @ 68°C
5. remove from PCR machine, pipet off wax, and chill briefly to re-harden thin wax layer
6. 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

• Mix DNA and Pfu by pipetting, then add wax.

1. 30" @ 95°C
2. 30" @ 95°C
3. 1' @ 55°C
4. 1'/kb @ 68°C
5. repeat steps 2-4 x 17 more times
6. 15' @ 68°C
7. hold @ 4°C