What are your comments regarding the longest size of a PCR amplified fragment, specially if I try to perform a multiplex PCR using as common one of the primers?
Sincerely, Rolando
phusion HF DNA poly #NEB M0530S is working really well with pretty high processivity
I've used it with 3 to 4kb fragment for cloning.
you should give more details in your question if you want the most accurate help (fgt size? origin of the DNA template ? what is for? etc
i also used the phusion DNA polymerase to amplify the long size fragments (around 7000bp), it worked pretty well, and the primers were just as normal, good luck!
For site directed mutagenesis , amplified an entire vector+insert of 6.6 Kb total size with pfu turbo .
I imaging multiplexing long amplicons is going to be more trouble than it is worth. Especially as it seems you are implying fragment lengths at the extreme end of long just by the nature of your question. The longer the fragments, the greater the opportunity for complementarity between you amplicons. I have never developed a multiplex assay, but I'm pretty sure a standard rules of thumb for any PCR reaction is, the smaller you can get away with designing your amplicons, while still maintain what is informative about the assay, the more efficient your amplification will be. I'm sure designing multiplex assays with small amplicons is challenging enough.
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