Q5 high fidelity dna polymerase

FUCCI plasmids were constructed starting from zebrafish mKO2-zCdt1(1/190) and mAG-zGem(1/100) plasmids, replacing the original promoter with the zebrafish ubiquitin promoter.
Original plasmids were amplified in E. coli and purified with Wizard^® Plus SV Minipreps DNA Purification kit from Promega. Then, 2 μg of each plasmid was cut with Nhe1 and BamHI, ran on an agarose gel, and the higher molecular mass band was purified using Wizard^® SV Gel and PCR Clean-Up kit from Promega.
Ubiquitin promoter was amplified by PCR from pENTR5′_ubi using Q5^® High-Fidelity DNA Polymerase, with these primers (F: 5′-cattgaGCTAGCatggatgttttcccagtcacgacg-3′, R:5′-tgactaGGATCCtgtaaacaaattcaaagtaagat-3′) and the following thermocycling protocol: 98 °C 30′′ (98 °C 10′′, 52 °C 30′′, 72 °C 2′) X35 cycles 72 °C 2′ pENTR5′_ubi was a gift from Leonard Zon (Addgene plasmid # 27320).
The PCR product was ran on an agarose gel and the band-purified using Wizard^® SV Gel and PCR Clean-Up kit from Promega.
Vectors and the ubiquitin promoter insert were ligated over night using NEB T4 DNA Ligase, in a molar ratio of 1:3, mixing 50 ng of vectors and 73 ng of insert.
The resulting plasmid was then amplified in E. coli and purified with Wizard^® Plus SV Minipreps DNA Purification Promega. This final plasmid was injected into 1-cell stage embryos together with the tol2 synthetic RNA.

The full-length mouse Arg2 3′UTR (270 bp) was amplified using Q5 High-Fidelity DNA Polymerase (NEB) and inserted into XhoI-digested pmirGLO vector (Promega) using the GenBuilder Cloning Kit (Genscript). Plasmids were isolated from bacterial cultures with the Plasmid Midi Kit (Qiagen, 12143) and the fidelity of the resulting construct (i.e., pmir_Arg2) was confirmed by sequencing. The sequences of cloning and sequencing primers are reported in Supplementary Table 2.
Raw 264.7 cells were seeded in a 96-well plate at a final density of 80,000 cells/well and incubated for 24 h. Cells were then co-transfected with 100 ng of pmir_Arg2 and either mirVana™ miR-155-5p mimic (25 nM, ID MC13058, ThermoFisher Scientific) or inhibitor (50 nM, ID MH13058, ThermoFisher Scientific) in serum-free DMEM using TransIT-X2 Dynamic Delivery System (Mirus) as transfection reagent. Luciferase activity was assessed at 24 hours after transfection using Dual-Luciferase Reporter Assay (Promega, E1910) according to the manufacturer’s instructions. RLU (relative luciferase units) expressed as mean value of the firefly luciferase/Renilla luciferase ratio of three independent experiments performed in triplicate were used for statistical analyses.