Muratore:Protocols/PCR/Quikchange

Basic QuikChange protocol

 * 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. It is especially helpful for large inserts.
 * 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:


 * 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