I wish I'd found this paper years ago. Table 1 has a straight-up list of golden gate overhangs that work well and play nice with each other, up to a 30-piece assembly. No more choosing four random bases and hoping.
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@ATinyGreenCell I had not. Most of my cloning doesn't fit into clean frameworks (dropping a barcode into a transposon right now), but I'll print this out and see if it's useful. Thanks!
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@StorkDevon @Eyesgack Great paper. I use the data to generate optimal overhangs when synthesizing dna -
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@StorkDevon ApE's golden gate designer tool uses the NEB 3 and 4 base dataset to design optimal compatibility of overhangs at each junction of a construct. You don't need to add the extra four bases or design by hand. It gives you a list of primers that you need too.
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@StorkDevon Yes, it’s helpful, but there is no reason to worry. If you choose overhangs that differ by 2 bases (check both ends) and are no palindromes, all overhangs will work for Golden Gate Cloning.
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@StorkDevon A bunch of other people have already recommended it but check out NEB's Golden Gate tool! You can put your overhangs in and it'll tell you the estimated number of incorrect/correct clones - no need to guesstimate in this day and age!!!
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