Take the 2-minute tour ×
Biology Stack Exchange is a question and answer site for biology researchers, academics, and students. It's 100% free, no registration required.

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?

share|improve this question
1  
Here is the original article. I'm going to get working on that OWW article. ncbi.nlm.nih.gov/pubmed/2235490 –  bobthejoe Jun 20 '12 at 8:58
add comment

1 Answer

up vote 1 down vote accepted

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.

share|improve this answer
    
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? –  shigeta Jun 24 '12 at 16:10
    
I think i discovered that part of the answer to this question is that Gibson assembly is patent protected... –  shigeta Apr 4 at 17:08
add comment

Your Answer

 
discard

By posting your answer, you agree to the privacy policy and terms of service.

Not the answer you're looking for? Browse other questions tagged or ask your own question.