I'm planning to join together two large fragments (a 10 Kbp vector and a 6 Kbp insert), and I'm wondering whether it's best to use Gibson Assembly or yeast-based homologous recombination. Has anyone used Gibson Assembly to join such large fragments? What's the largest you've ever joined?
A guy named Daniel Gibson used it to assemble an entire synthetic mycobacterium genome http://www.ncbi.nlm.nih.gov/pubmed/19363495. So, length by itself is not the limiting factor. Length just makes it more likely that you'll have other limiting factor somewhere in it, like secondary structures...
Hi Fabrizio,
I think John is right. We frequently use the Gibson Assembly Master Mix to assemble strands, but never tested such long strands.
Maybe you also could increase the temperature above the recommended 50° C. I guess that this will not affect the efficiency of the assembly reaction (Taq DNA ligase and Q5 polymerase are thermostable) but decrease the probability for the formation of limiting secondary structures.
Best regards,
Manu
Thanks Manu. I know the protocol says the kit has been tested for up to 20 Kbp, but other than using specific competent cells they don't recommend anything else for the actual reaction. I'll try increasing the temperature to avoid secondary structures though, as the fragment I'm using has high GC content.
Did it work out? I am assembling 7.7kb and 4.5kbp and so far it has not really worked.
Hi Marta, I've also tried few times, changing conditions, but in the end I went for yeast-based homologous recombination, as my vector had features for replication and selection in yeast. Sorry I can't help you further and good luck.
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