Does anyone have any TOPO directional cloning tips? This question is board and essentially asking how to clone. However, it is practical and answerable but may be falling into a discussion. I was curious how to constraint what is a very well meaning question?
I agree at the closure of this question within the format of stack ex, however to say that it will solely have answers backed by opinion I believe is incorrect.
I find that using Pfu polymerase gave a particularly good yield of blunt ended PCR product which should be used in downstream TOPO cloning. [See agarose gel of PCR product]. I also tried NEB Phusion polymerase with drastically inferior yields [see agarose gel of poor yield]. So my tips is to use Pfu Polymerase.
I appreciate this question could be phrased as "which polymerase is best suited to directional TOPO cloning?" however I wanted to invite a broad range of insights before I begin the experiment so I am fully aware of problems or points of care that I should be aware of before I begin.
I closed the question now, I don't think asking for general tips with a method is a type of question that works well with the Q&A format. Very general questions whithout right answers and possibly a lot of different, equally valid answers are problematic on SE sites. But such a question is not far from being a constructive and useful question.
Several very similar questions would be perfectly fine for the site, the most obvious cases would be if a user runs into any kind of problem with that method and then asks for help solving that problem.