How many nucleotides can be placed between the overlap and annealing section of the final reverse primer for NEB assembly (Gibson assembly)? P/T2A ok? This would be the last reverse primer that overlaps with the vector backbone.
I think this is more limited by the generation of the PCR fragment than the assembly reaction itself. In my experience PCR starts to produce less and less specific product over a certain primer length, with any primers over 50-60 bp becoming very iffy. Since you need ~20 bp for overlap, and ~20 bp for the specific 3' end of the primer, this realistically limits your spacer to around 10-20 bp per primer. However, you can also get a spacer oligo with overlaps to the ends of your fragments synthesized which can then be integrated into the vector during the assembly reaction. Since it doesn't need to be used as an actual primer you have a lot more freedom with the length and sequence.
Ah, so if you get the correct pcr product, then you could have a longer spacer? I have already performed the Pcr with a 100 mer and get a clear single band of correct length.
Yeah as long as the primer works and it has an overlap on the 5' end that will cause assembly I don't think there's really a limit.
Great, thanks for your feedback! Now I just have to miniprep and screen screen screen!
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