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TALEs, TALENs and ICA (iterative capped assembly)

January 22, 2013

So I’ve spent the last months working on/with TALEs (transcription activator-like effectors).  (Epi)Genome is going to be the sport of the future (like kickboxing – 80’s movie reference).  Anyway, there are a bunch of TALE assembly platforms out there.  I decided to go with ICA because of the flexibility of incorporating any RVD.  The protocol works but it appears many have struggled with getting it to work.  The paper doesn’t go into the technical details very much or highlight the critical steps so I thought it would be helpful to some to post a detailed version of the protocol exactly the way I am doing it.  One thing I will note here is that production of the monomers, while it is simply a PCR and restriction digest is not trivial.  I had difficulty getting good PCR products and more importantly BsmBI is not a good enzyme.  You will have to use more enzyme and cut longer then you think.  You may even have to gel purify away the uncut/partially cut monomers.  I’m also using a lot less monomer than what Adrian Briggs is using in his protocol.  This is nice as it saves you from the time and cost of making new batches of monomers.  I would also suggest doing the cloning part of the protocol the old fashion way (linearize and phosphatase treat the vector) and not doing a Golden Gate reaction.  I’ve read far too many posts of people struggling with Golden Gate cloning of TALEs.  Once it is working then, you could try to go with Golden Gate.

On another note – I probably should have posted this protocol a few months ago as it appears that there is a much easier technology coming on-line.  CRISPR

But for those still interested in making TALEs the protocol is here: TALE construction protocol – Iterative Capped Assembly

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3 Comments
  1. mc1061dm1 permalink

    Hi, Ethan,

    Have you tried to optimize the PCR library amplification step? My starting material is limited, and need ~20-25 cycles to amplify. I was wondering whether a higher efficiency of PCR can decrease the cycles needed.

    Thanks

    Ming

    • ethanomics permalink

      Hi Ming,
      I’m not sure exactly what you are referring to as this is a post about TALE assembly and it seems like you are talking about NGS library amplification. Anyway, assuming you are talking about NGS library amplification, 20-25 cycles in my experience is too many and produces ugly data with too many PCR duplicates. Perhaps not worthless data but definitely not pretty data. Higher efficiency PCR would decrease the number of cycles needed. How to get higher efficiency PCR is the problem. I would definitely use the Kapa HF polymerase. It is significantly better then the TruSeq polymerase. Actually, I think the best thing to do when you get a TruSeq kit is throw away the polymerase. There may be some other things to do but they would be application specific.

  2. mc1061dm1 permalink

    Sorry for not specific.

    I AM referring to NGS library. Have you played with enzyme amount, annealing temp, etc to improve the efficiency?

    Thanks

    Ming

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