Spacers of homologous recombination primer design

[manulera]

Respect to what does it make sense to define them?

• With respect to the insert, so that if the insert is inverted it goes the other way around
• With respect to the homology region (as is) so that they are always read in the same direction

[dgruano]

My two cents would be to define them with respect to the insert:

• Ontologically, the spacer is also inserted.
• Practically speaking, you can perform a PCR and obtain product that has insert + spacer + recombination tail, and stop it there.
• Mostly, the applications that come to my mind of adding a spacer, it is an addition to the insert (e.g. when adding a GFP construct from a plasmid to a random genetic locus, if you want to add a small tag at the C-ter end, you can do it with either of the recombination tails, but always with the C-ter-annealing primer).

I may be missing something on how the primer design works, so let me know if what I said makes sense.

[manulera]

Thanks for the thought @dgruano

I agree that it seems to make sense in this case, but for the gibson assembly it only makes sense to define the spacers with respect to the final assembly (regardless of whether each fragment is oriented in either direction), so the spacer would have a different meaning in each case

[dgruano]

Ha! It's a tricky call when you introduce the Gibson Assembly indeed...