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Ligase Independent Cloning is a protocol that allow an insert to be integrated into a vector without ligation. It uses T4 DNA polymerase with only ATP to first chew back from blunt ends to create long sticky ends and then a polymerase treatment with full dNTP compliments to fill in the vector.

While there are several nice articles and resources that describe the procedure, I really would like a protocol with concentrations, temperatures and timing, which I'm having problems finding. OpenWetWare for instance has only stub pages with no details. Can anyone point to a full step by step recipe to make this work, once you have designed the primers?

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You can look up Gibson Assembly or Circular Polymerase Extension cloning (CPEC). For both of these the website for J5 has some good protocols. Here is the one for CPEC: http://j5.jbei.org/j5manual/pages/80.html

For CPEC you can look at the 2011 Quan paper: http://www.ncbi.nlm.nih.gov/pubmed/21293463

Hopefully that helps.

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  • $\begingroup$ thanks for this @Justin, but the protocol link, while complete doesn't make clear to me how this works. Why does the Pfusion polymerase have all the dNTPs? I had thought that to chew back from a blunt end, the polymerase would need an incomplete set of NTPs? Also how is the vector cut? Is there any decision to be made here or is it just any digestion cut at all? $\endgroup$
    – shigeta
    Commented Jun 24, 2012 at 16:10
  • $\begingroup$ I think i discovered that part of the answer to this question is that Gibson assembly is patent protected... $\endgroup$
    – shigeta
    Commented Apr 4, 2014 at 17:08

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