Phusion U Multiplex PCR Master Mix

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

Generation of bacterial TadA* libraries (evolution rounds 1–3, 5, and 7)
Briefly, libraries of bacterial ABE constructs were generated by two-piece USER assembly of a PCR product containing a mutagenized E. coli TadA gene and a PCR product containing the remaining portion of the editor plasmid (including the XTEN linker, dCas9, sgRNA, selectable marker, origin of replication, and promoter). Specifically, mutations were introduced into the starting template (Supplementary Table 7) in 8 × 25 μL PCR reactions containing 75 ng-1.2 μg of template using Mutazyme II (Agilent Technologies) following the manufacturer’s protocol and primers NMG-823 and 824 (Supplementary Table 6). After amplification, the resulting PCR products were pooled and purified from polymerase and reaction buffer using a MinElute PCR Purification Kit (Qiagen). The PCR product was treated with Dpn1 (NEB) at 37°C for 2 h to digest any residual template plasmid. The desired PCR product was subsequently purified by gel electrophoresis using a 1% agarose gel containing 0.5 μg/mL ethidium bromide. The PCR product was extracted from the gel using the QIAquick Gel Extraction Kit (Qiagen) and eluted with 30 μL of H2O. Following gel purification, the mutagenized ecTadA DNA fragment was amplified with primers NMG-825 and NMG-826 (Supplementary Table 6) using Phusion U Green Multiplex PCR Master Mix (8 × 50 μL PCR reactions, 66 °C annealing, 20-s extension) in order to install the appropriate USER junction sequences onto the 5’ and 3’ end of the fragment. The resulting PCR product was purified by gel electrophoresis. Next, the backbone of the bacterial base editor plasmid template (Supplementary Table 7), was amplified with primers NMG-799 and NMG-824 (Supplementary Table 6) and Phusion U Green Multiplex PCR Master Mix (100 μL per well in a 98-well PCR plate, 5–6 plates total, Tm 66 °C, 4.5-min extension) following the manufacturer’s protocol. Each PCR reaction was combined with 300 mL of PB DNA binding buffer (Qiagen) and 25 mL of the solution was loaded onto a HiBind DNA Midi column (Omega Bio-Tek). Bound DNA was washed with 5 column volumes of PE wash buffer (Qiagen) and the DNA fragment was eluted with 800 μL of H2O per column. Both DNA fragments were quantified using a NanoDrop 1000 Spectrophotometer (Themo Fisher Scientific).

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