Can I clone an oligonucleotide/shRNA oligonucleotide using In-Fusion Cloning?
Can I clone an oligonucleotide/shRNA oligonucleotide using In-Fusion Cloning?
Yes, synthetic oligonucleotides (?50 bp), carrying homologous overlaps with the termini of the linear vector, can be cloned using In?Fusion Cloning. For cloning of short synthetic oligos (between 50 bp and 150 bp), the suggested oligo-to-vector molar ratio is 5–15:1. Depending on the oligo length, the optimal molar ratio must be determined empirically.
Note: Non-phosphorylated oligonucleotides are compatible with In?Fusion Cloning. However, 3' exonuclease activity in the In?Fusion enzyme mix requires terminal 3' OH groups.