We recently had a tech that passed through who ordered Q5 for her mutagenesis PCRs. She clearly had things working because she created 10-20 constructs that I checked the sequences for. However, she is no longer here, there's a ton of Q5 left, and her notebook is quite vague. I've only used Phusion for mutagenesis, and cannot seem to get the Q5 to work in a similar fashion. I see that the kits sold by NEB include a KLD enzyme kit, but I don't think she was using that, or at least I can't find it anywhere. Our tech is trying to use the Q5 but getting no colonies after incubation with DpnI and transformation. The primers are complimentary with the desired mutation. Is there something that I'm missing?
Thanks!
Are you using the NEB Tm calculator for setting up annealing temperature? Something about Q5 polymerase and their buffer makes it so that the optimal annealing temperature is higher than with most other polymerases. I've gotten mutagenesis to work fine with Q5 as long as I've used the calculator first, though in some cases it just meant doing annealing and extension together at 72C.
I didn't need to change anything about the downstream DpnI digest, that was identical to the normal protocol.
We contacted NEB and figured things out. Q5 requires much higher template concentration if you don't use their kit. We ran a bunch of different conditions and used a two step cycling program and everything is working now. Thanks!
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