Hi all,
I have been struggling to linearize an 8.4 kb vector by PCR for subsequent Takara Infusion cloning. I am currently using Phusion polymerase with the following protocol
98 C - 30 sec
98 C - 15 sec
60 C - 30 sec
72 C - 4min30sec
Repeat 2-4 34x
72 C - 5 min
Hold at 4 C
Template DNA - 5 ng
I have also used a 64 C annealing temperature (which was the computed optimal annealing temp for my primers) and still no amplification. Have also tried +/- DMSO but still no products. Template has been sequence verified and primer set has been used successfully to amplify a smaller 7 kb vector. Primers do anneal right next to one another but as previously mentioned, this was not a problem for the smaller vector.
Any advice would be appreciated. Happy to provide additional clarification.
I would try using the gradient temperature feature of the thermocycler. Also I have used Gibson assembly to ligate smaller fragments of such a large fragment, as I have also had problems with PCR and fragments past 6kb length.
Just tried a gradient PCR with annealing temps ranging from 55-65 C. No luck with the amplification.
I would now clone the 8.4 fragment into two 4.2 kb fragments and join them with scarless Gibson assembly in an easy one pot reaction after you PCR the fragments with primers designed to have assembly overhangs here https://nebuilderv1.neb.com/
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