Hi all. I've been trying to use the Agilent QuikChange XL Site-Directed Mutagenesis Kit, and haven't been getting any PCR product on a gel after performing DPN digestion.
My primers were designed using http://www.genomics....signProgram.jsp
GC content ranges from 48% to 54%, melting temperature is greater than 78C using the agilent equation, and length ranges from 46-54 bases. Primers are complimentary, as per the Agilent protocol.
I'm trying to make five different mutants, each separately, each with 5 bases substituted, and none of the 5 primer pairs has worked. Since it's not just one pair of primers failing, and they were all designed using the recommended program, I'm skeptical that primer design is the problem.
My plasmid is about 5kb in size.
My standard reaction uses
5ul 10x reaction buffer
125 ng of each primer
1ul of dNTP mix
3ul of quicksolution
1ul of pfuTurbo polymerase
ddH2O to bring to 50ul
for template I've tried 5, 10, 20, 50, and 100 ng (while maintaining 125 ng of each primer)
I've also tried adding 1.5uL of DMSO
I've used the recommended cycling parameters:
* 1 cycle of 95C for one minnute
* 18 cycles of 95C for 50 seconds, 60C for 50 seconds, 5.2 minutes of 68C
* 1 cycle of 7 minutes of 68C
I've tried using fresh dNTPs and new pfuTurbo polymerase. Switching to a different kit isn't really an option (we have several of the Quikchange kits).
Any and all assistance is appreciated.
Edited by MaryNelson, 13 May 2015 - 01:45 PM.