I'm planning to use Golden Gate cloning in order to insert guide RNA spacer sequences into a cloning vector between two head-to-head BsaI restriction sites. Prior to setting up the reaction with my vector, 5' phosphorylated dsDNA oligonucleotides, BsaI, and T4 ligase that I will set on a thermal cycling program according to Engler 2009, should I first digest the destination vector with the BsaI enzyme alone and then gel purify the backbone fragment?

Likewise, should I desalt my dsDNA oligos after phosphorylation and annealing, but before adding them to the Golden Gate setup? What would be a good kit or protocol to do this without losing 21-25 bp fragments?

And finally, has anyone found a specific molar ratio for gRNA insert to backbone that is more efficient for Golden Gate cloning or should I just stick with the 1:1 ratio used for cloning larger inserts?

Any and all advice is appreciated