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I'm designing a gene in DNAWorks, and I'm unable to find a set of conditions that gives an "overall score" of zero (which is what I normally insist on). People that have used DNAWorks: what's the highest nonzero score you've been able to successfully assemble?

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Can you give us more information on what you are trying to assemble (size, length of overlap, length of oligos) and how you plan to assemble it? –  Gergana Vandova Jun 14 '12 at 0:24
    
The gene is ~600 bp in size. I'm flexible on the oligo length and overlap length, but I was thinking something like 50-60 bp oligos and 15 bp overlaps. I'll assemble it by Gibson assembly directly into the vector backbone, but the part is designed so that I can also assemble it the standard way: first assembling the gene by PCR and then ligating it into the vector. Thanks! –  Kunal Jun 14 '12 at 19:17
    
@GerganaVandova, I think that the question is trying to get at the design portion and not the actual building portion of gene assembly. –  bobthejoe Jun 15 '12 at 6:46
    
@bobthejoe I am trying to say that the design portion is not the bottleneck. –  Gergana Vandova Jun 15 '12 at 7:11
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1 Answer 1

You might want to read Gibson's paper on the step-wise assembly of the mouse mitochondrial genome (1):

enter image description here

He started with 60b long oligos with 20b overlap, as he assembled 5 of those 60-mers into a backbone, obtaining 384b fragments. On the next step, he joined 5 of those 384mers, obtaining 1.2kb constructs. You can do the same, but on the second step use 2x 384mers to get you ~600bp gene. Gibson didn't use DNAWorks to chop up the sequence. He just started from base 1, so that his fragments were F1 [1:60], F2[41:100], F3[81:140], etc.

I think that 15b homology is pretty low (Gibson used 20b for assembling the 60mers in the first step, and 40bp homology for the next assemblies). 60b oligo length is standard.

You can also try one step assembly with all of your oligos (via PCA) as you planned, but I think that the two step assembly might be more efficient.

Let me know if you need more help.

1. Gibson et al, 2009. Chemical synthesis of the mouse mitochondrial genome

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