Pgem t easy sub cloning vector

Depending on the arrangement of the exons, the first 1–3 and last 1–2 exons of all 11 S. mansoni PDEs were amplified from genomic DNA, using primers upstream or downstream of the predicted exons and a primer in the predicted exon (see S4 Table for primers and details), and a proof reading polymerase (Phusion; NEB, Hitchen, UK). This sequence information was used to design primers to amplify the complete coding sequence for each PDE from mixed adult worm cDNA, again using Phusion polymerase. Some PDEs were amplified in two sections due to size/primer incompatibility (S4 Table). These strategies were successful for all predicted SmPDE ORFs except SmPDE2. All sequences generated were submitted to GenBank (Table 1). cDNA produced from different S. mansoni life-cycle stages (schistosomula; juvenile female/male worms; adult female/male worms) were used in attempts to amplify the complete SmPDE2 or sections thereof, alongside attempts to amplify individual exons from genomic DNA. All amplified fragments were ligated into the pGEMT-Easy subcloning vector (Promega, Southampton, UK) and Sanger sequenced by Source BioScience (Nottingham, UK). It should be noted that a recently launched online resource schisto.xyz [37 (link)] lists SmPDE2 as detectable by RNAseq, and expressed particularly in adult worms (SmPDE2).

Genomic DNA (gDNA) was isolated from either sperm or tail samples, using the GenElute Mammalian Genomic DNA MiniPrep Kit (Sigma). For sperm samples, the mirVana RNA extraction lysis buffer treatment was performed prior to this extraction (Life Technologies). After gDNA was isolated, 1 μg was bisulfite treated, following the instructions with the NEB Epimark Bisulfite Conversion Kit (NEB). Following cleanup, bisulfite-specific PCR amplicons were amplified using GoTaq 2X, covering the promoter region (Promega). These PCR products of 170 bp. were run on 2% agarose gels, and the bands of interest were isolated and gel purified with the Qiaquick PCR purification kit (Qiagen). The resulting DNA extracts were cloned into the pGEM T-easy subcloning vector (Promega), and 2 μl of ligation reaction was then transformed into 50 μl of 5α competent cells (NEB), and grown overnight on ampicillin-LB-Agar plates at 37C (NEB). Individual colonies were grown in ampicillin liquid culture and their DNA was extracted using the Zyppy plasmid Miniprep Kit (Zymo Research). Plasmid DNA was then digested with EcoRI (to confirm the insert) and sequenced, using the SP6 sequencing primer. Sequencing was performed at the Nevada Genomics Center. Individual CpGs were then quantified for bisulfite conversion and the totals were calculated.

For StARF10-6×His protein preparation, the coding sequence of StARF10 was PCR amplified and cloned into the pET28a⁺ vector (Novagen). Protein expression was performed using Escherichia coli BL21(DE3) cells as host, followed by Ni-NTA affinity column-based protein purification. For bait DNA preparation, promoter fragments P1, P2, and P3 of prom:StGH3.6 were PCR amplified from potato genomic DNA, and promoter fragment P4 of prom:AtGH3.5 was PCR amplified from Arabidopsis genomic DNA. All the fragments were cloned in the pGEM-T Easy sub-cloning vector (Promega) and their sequences were verified. For the electrophoretic mobility shift assay (EMSA), probes were prepared by labeling promoter fragments with γ-³²P-ATP using a KinaseMax End-Labeling kit (Ambion). The binding reactions were carried out as described previously (Chen et al., 2004 (link)).