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Has anyone tried the chew back-anneal in vitro DNA assembly method (known as Gibson Assembly) for difficult sequences, like GC-rich sequences? How big constructs could you efficiently assemble? Did you use the original protocol or some optimizations?

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Has anyone heard of Gibson Assembly? –  Gergana Vandova Dec 14 '11 at 22:29
    
I've just joined the Gibthon team, so if you need any more specific help let me know. –  Richard Smith Sep 3 '12 at 10:10

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I haven't tried Gibson Assembly myself, but I do know the Cambridge iGEM Team in 2010 and 2011 developed a web-based tool - Gibthon - for automatically generating primers for Gibson Assembly.

You could try running your difficult sequences through their tool to see what it suggests for primers. It apparently gives you a "value for the Gibbs free energy of the worst case secondary structure of the primer."

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Thanks. Yes, I have heard of the Gibthon software, haven't tried it yet. We have been using the IDT predictor of secondary structure of the primers. Might be worth comparing it to the Gibthon tool. It would have been nicer if some optimizations of the reaction conditions make the use of such predictors obsolete. Not to mention that their precision is questionable. –  Gergana Vandova Dec 14 '11 at 22:55

I also haven't tried it, but theoretically GC-rich sequences will interfere with Gibson assembly. Since unique homology between the ends is key to the assembly process, repetitive sequences which cause non-unique overhangs will increase the possibility of incorrectly ordered assembly. One solution would be to use alternative codons where possible to decrease repetitiveness.

I'm sure all the overlap design tools will try to avoid repetitive overlaps. Prof Jim Hasselhoff at Cambridge Plant Science is a big proponent of Gibson assembly, it was one of his students who wrote Gibthon. If you email him I'm sure he'll give you specific advice.

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