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fusion of tow PCR products
govind kunduri
Tuesday, 11 September 2007 15:38 UTC
hi
I want a protocol for fusing two PCR products (A and B)which resulting in one continuous PCR product(A-B).
Can any body suggest how to perform this on the basis of PCR without ligation step.
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Replies
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We routinely use fusion PCR to fuse 2-6 fragments. We have published a detailed protocol for fusing three fragments (Nature Protocols volume 1 P3111 and following). Two fragments is marginally easier. To obtain consistent results it is important to follow the procedure closely (thermal ramp rates, enzymes, melting points of primers, etc.) The only change we have made since the publication is that we sometimes use Hot Start KOD polymerase for the fusion reaction because it has stronger proofreading activity than Accuprime Taq High Fidelity. If KOD is used, the annealing temperature for the fusion PCR should be as specified in the KOD brochure. Other aspects of the protocol are unchanged.
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Hi Dr Edgerton, I very interesting in your paper:Fusion PCR and gene targeting in Aspergillus nidulans. Is possible that you send me a PDF copy? I really appreciate it
Thanks
Bernardo Gonzalez
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Hi Bernardo,
If you are interested in the fusion PCR aspect of Dr Edgerton’s paper in Volume 1 of Nature Protocols, you may be interested to know that Volume 2 is currently free to access (until the end of October 2007), and contains a similar protocol provided by Dr Karin Heckman and Professor Larry Pease: Gene splicing and mutagenesis by PCR-driven overlap extension.
If you do use either of the protocols and have feedback that would be useful to other potential users, please do post a comment via the abstract page of the protocol. An example of a Nature Protocol where comments have been posted can be found here.
Good luck with your experiments!
Best,
Dot
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