Wizard sv gel and pcr clean up system

Genomic DNA samples were obtained from liver tissue from male and female individuals of Pseudis and Lysapsus species (Supplementary Table 1) following the TNES method employed by Medeiros et al. (2013) (link). Integrity of genomic DNA was evaluated by electrophoresis in a 0.8% agarose gel and total genomic DNA was quantified in a Nanodrop (Thermo Fisher, United States) spectrophotometer. PcP190 satDNA was isolated by PCR using the primers P190F (5′-AGACTGGCTGGGAATCCCAG-3′) and P190R (5′-AGCTGCTGCGATCTGACAAGG-3′), described previously by Vittorazzi et al. (2011) (link). The PCR program used was: (1) 94°C for 8 min; (2) 39 cycles of 94°C for 30 s, 58°C for 1 min and 72°C for 2.5 min; (3) 72°C for 8 min. PCR amplicons were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) and inserted in a plasmid pGEM-T Easy Vector (Promega). Plasmids were cloned into E. coli JM-109 bacteria using the TransformAid Bacterial Transformation Kit (Fermentas), according to manufacturer instructions. Recombinant colonies were selected, plasmid DNA was extracted following Sambrook et al. (1989) , and the inserts were amplified by PCR using the T7 and SP6 universal primers. After purification with the Wizard SV Gel and PCR Clean-Up System (Promega), the cloned fragments were sequenced on both strands using BigDye Terminator (Applied Biosystems) according to manufacturer instructions.

For the cloning of the WT and mutant exon12 of FPGS into pGL3-basic luciferase reporter vector (Promega), WT exon12 was amplified from CCRF-CEM cDNA and the mutant exon12 was amplified from MTA C-3 cDNA using the following primers: NheI/Exon12 Fw (5′-TAATGCTAGCTTCGGAACACGGAGTG) and Ex12/BglII Rv primer (5′-TAATAGATCTCGGCCTCTCGCGG). PCR products were purified using Wizard® SV Gel and PCR Clean-Up System (Promega) and cut along with the pGL3-basic vector using the restriction enzymes NheI and BglII (NEB). For the cloning of a 150bp fragment from intron 14, PCR was performed on genomic DNA from MTA C-3 using the primers SmaI/intron14 Fw (5′-ATCTCCACCTCCCGGGTTC) and Int14/DpnII Rv (5′-GGCGGATCACCTGAGGTCAA). The PCR product was first resolved on a 2% agarose gel and purified using Wizard® SV Gel and PCR Clean-Up System (Promega), and then it was excised with SmaI and DpnII (NEB, Ipswich, MA, USA). Ligations were performed using DNA ligation kit (Takara, Otsu, Shiga, Japan) according to the manufacturer's instructions.
For the luciferase assay, MTA C-3 cells were electroporated with 10μg of each vector together with 0.2μg of pRLO-Renilla vector (0.234kV). Thirty-six hours following transfection, luciferase activity was determined using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions on a GloMax 20/20 luminometer (Promega).

The Tol2 mRNA was transcribed from the pCS-TP plasmid whereas eGFP-Ireb2-3′utr and standard fluorescence control mRNAs were transcribed from plasmids: N.f CMV/SP6: eGFP-Ireb2-pA, D.r CMV/SP6: eGFP-Ireb2-pA and CMV/SP6: RFP-pA, respectively. All were previously linearized using NotI and purified with Wizard® SV Gel and PCR Clean-Up System (Promega). Then, 1 μg of each linearized plasmid was transcribed using the mMESSAGE mMACHINE SP6 kit (Ambion) according to the manufacturer’s protocol. For DIG-labeled probe synthesis, initially sequences were amplified by PCR from cDNA using a reverse primer carrying a T7 promoter sequence on its 5′ end; 200 ng of PCR product, previously purified with Wizard® SV Gel and PCR Clean-Up System (Promega), were directly transcribed using T7 enzyme (Fermentas) and digoxygenated RNTP mix (Roche) for 2 hours at 37 °C. The in vitro transcription mix was precipitated with 1/10 of volume of LiCl (5 M) and 2.5 volumes of isopropanol, than washed with 70% ethanol and finally resuspended in nuclease-free water and stored at –80 °C.

The complete sequence of p29 (795 nts) and partial sequence of p32 (288 nts) in the RNA1 and RNA2, respectively, of CiLV-C isolates, were obtained using described primers (Locali et al., 2003 (link); Ramos-González et al., 2016 (link); Table 2). Amplicons were obtained from 26 samples collected in non-commercial citrus regions in Brazil and Argentina, from 2006 to 2019, and 31 from commercial citrus orchards inside the citrus belt SP-MG, in the period 2017 to 2019 (Supplementary Table 1). After RT-PCR, amplicons were purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, United States), and cloned into pGEM-T-Easy (Promega, Madison, WI, United States). Plasmids were transformed into Escherichia coli DH10β competent cells by electroporation, and 5–10 recombinant clones derived from each sample were sequenced by the Sanger method (Instituto Biológico, SP, Brazil). PCR products from some samples collected in non-commercial orchards (Supplementary Table 1) were directly sequenced after the purification using the Wizard SV Gel and PCR Clean-Up System (Promega).

To determine the mutated allele, a small piece of tail was cut from a single F2 heterozygous progeny (of each allele) to extract genomic DNA through incubation at 98°C for 20 min in 40μl 25mM NaOH in a 96-well plate, then neutralized with 40μl of 40mM Tris-HCl. The PCR amplicons were amplified using Takara Ex Taq DNA Polymerase (Takara Bio, RR001A), purified with the Promega Wizard SV Gel and PCR Cleanup System (Promega, A9282), and examined on a 1% agarose gel (for examining alternative splicing) and sequenced by the UAB Heflin Center for Genomic Sciences Sanger Sequencing Core.