Q5® Hot Start High-Fidelity 2X 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.	Always thaw on ice

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

Publication protocol

General methods

DNA amplification was conducted by PCR using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2× Master Mix (New England BioLabs) unless otherwise noted. All mammalian cell and bacterial plasmids generated in this work were assembled using the USER cloning method as previously described39 and starting material gene templates were synthetically accessed as either bacterial or mammalian codon-optimized gBlock Gene Fragments (Integrated DNA Technologies). All sgRNA expression plasmids were constructed by a 1-piece blunt-end ligation of a PCR product containing a variable 20-nt sequence corresponding to the desired sgRNA targeted site. Primers and templates used in the synthesis of all sgRNA plasmids used in this work are listed in Supplementary Table 5. All mammalian ABE constructs, sgRNA plasmids and bacterial constructs were transformed and stored as glycerol stocks at −80°C in Mach1 T1R Competent Cells (Thermo Fisher Scientific), which are recA−. Molecular Biology grade, Hyclone water (GE Healthcare Life Sciences) was used in all assays and PCR reactions. All vectors used in evolution experiments and mammalian cell assays were purified using ZympPURE Plasmid Midiprep (Zymo Research Corportion), which includes endotoxin removal. Antibiotics used for either plasmid maintenance or selection during evolution were purchased from Gold Biotechnology.


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Manufacturer protocol

Download the product protocol from New England BioLabs for Q5® Hot Start High-Fidelity 2X Master Mix below.

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