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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
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.
- 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
|
- 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
- hold @ 4°C