Muratore:Protocols/PCR/Quikchange

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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.
  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). 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

References

  1. Wang & Malcolm (1999) - Biotechniques 26 680
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