Wizard plus sv minipreps dna purification kit

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.

Primers were designed to amplify fragments of TcLEC2, EGFP, and the vector backbone (pBin19) (Table A in S1 File). Genomic DNA was extracted as previously described using a CTAB extraction protocol [29 (link)] from non-transgenic cacao and the transgenic TcLEC2-GR tree. Purified plasmid DNA was generated for pGZ13.0313 using Wizard Plus SV Minipreps DNA Purification kit (Promega) and diluted to 5 ng/μl with salmon sperm DNA, a 20 μl PCR reactions were setup using Phusion Polymerase (New England BioLabs (NEB), Ipswich, MA) and 5 μM primer (Table A in S1 File) for: 94°C for 2 min, then 35 cycles of 94°C for 30 sec., 50°C for 45 sec, 72°C for 3 min followed by incubation at 72°C for 7 min. In a preliminary experiment, we observed the formation of a heteroduplex dsDNA consisting of the endogenous and transgene TcLEC2 products that annealed, thus the experiment was repeated with the addition of 5 μl of T7 Endonuclease I (NEB) to each reaction, followed by a 5 min incubation at 37°C to cleave the heteroduplex. Four μls of each PCR reaction were then loaded onto a 1.5% agarose gel (IBI Scientific, Peosta, IA) for electrophoresis.

Plasmids were purified using the Wizard^® Plus SV Minipreps DNA purification kit (Promega, Madison, WI, USA).