No PCR product in site-directed mutagenesis - (Apr/12/2012 )

I am introducing single amino acid changes into a 7.5kb vector using Pfuultra II fusion HS polymerase. I am trying to make 10 mutations, so far I have gotten 4 to work but the remaining 6 do not give a product. Here is the protocol for the 4 that worked.

50 µl PCR reaction

10X pfu reaction buffer 5 µl
10 mM dNTP mix 1.25 µl
Forward mutagenesis primer (100uM) 1.5 µl
Reverse mutagenesis primer (100 uM) 1.5 µl
dsDNA template pET-3Cpro (300 ng) 1.0 µl
pfu Ultra fusion II polymerase 1.0 µl
H₂O 38.75 µl
TOTAL 50 µl

Thermocycler profile

95ᵒC – 4’
95ᵒC – 30 sec
50ᵒC – 1’ X 25
72ᵒC – 16’
72ᵒC – 7’
4ᵒC - ∞

Many of the primers I use are long (35-42 bases) and have high Tm's 65-74C. I feel like I shouldnt lower the annealing temp any further since it is already down to 50C. I have tried 68C for the extension and 1 mutation was successful. I am really looking for some insight on PCR conditions for these reactiions. Any advice would be greatly appreciated

Thanks

How did you design your primers? They should be long, I had 40 and 45, the Tm will be always high. Did you check for secondary structures in your primers?

I bought a Stratagene Agilent Quickchange kit, and used the design tool that they provide. I got Tm's higher than 78 (calculated by their formula), and had annealing at 55. Extension at 68, with 1 min/kilobp. They recomended using exactly 125 ng of each primer for different ratios 5-50ng of vector template. But they transform it right away in the kit. It worked for me on two mutants on a first try.

You can have a look at their manual and see if there isn't anything usefull. They've got the guidelines for the primer design there too, I guess those are quite general.

Thanks for the tips Trof. I used primerX webtool to design the primers. I have used it in the past and have not had problems for site-directed mutagenesis. I repeated using annealing temp of 57. I am using much more template and primer than recommended so if the different temps do not work I can drop those amounts.

I heard lots of people saying some mutations work and others not, even with the same vector, even with kit, so I guess it's always depends on the sequence of primers and vector.
If you didn't try that yet, maybe try to increase the anneling or add some additives like DMSO or.. what was it some people swear on.. betaine? I think.

Trof on Fri Apr 13 14:43:33 2012 said: