Q5 hot start high fidelity polymerase m0494s

Using non-degenerate primers (Table S1), each sublibrary underwent PCR to generate linear DNA with overlapping regions between the VRs. Q5 Hot Start High-Fidelity polymerase (M0494S, New England Biolabs, Ipswich, MA, USA) was used in an overlap PCR for 18 cycles with an annealing temperature of 60°C to generate combined sublibraries. This was performed two at a time (i.e., A + B = AB; C + D = CD; AB + CD = ABCD; ABCD + E = ABCDE). After every overlap PCR, DNA products were run on a 1% agarose gel and gel purified using a gel DNA recovery kit (D4007, Zymo Research, Irvine, CA, USA). This parental library was digested, purified, ligated, transformed, and subjected to large-scale plasmid isolation identical to the methods described above in the “Sublibrary Plasmid Construction” section. This product was considered the parental plasmid library.

Degenerate PCR primers (see Table S1) were ordered (Eurofins Genomics, Louisville, KY, USA) and PCR was performed using the wild-type AAV2 cap gene from pSub201 as a template and Q5 Hot Start High-Fidelity polymerase (M0494S, New England Biolabs, Ipswich, MA, USA). 25 cycles using the suggested parameters on NEB’s website were followed. PCR products were run on a 1% agarose gel and gel purified using a gel DNA recovery kit (D4007, Zymo Research, Irvine, CA, USA).