Minibest dna fragment purification kit ver 4

Ctenopharyngodon idella enteric DNA was extracted using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0; the Pept1 gene promoter was cloned using Genome Walking Kit, and the PCR product was purified using TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0. The promoter of the Pept1 gene was cloned using Genome Walking Kit. The pCMV-N-Flag and PGL3-basic (Promega) vectors were digested with BamHI and XhoI and purified using a plasmid purification kit (Magen); JAK2 from C. idella and the promoter of the Pept1 gene with BamHI and XhoI double restriction sites were purified. The fragments were ligated overnight and transformed into competent DH5α cells. Positive clones were selected. After successful sequencing, cultures were expanded, and the pCMV-N-Flag-JAK2 and PGL3-basic-Pept1 recombinant plasmids were purified using a high-copy low endotoxin plasmid extraction kit (HiPure Plasmid EF Midi Kit, Magen).

Plasmids used to transfect P. falciparum were prepared based on the Multisite Gateway System (Thermo Scientific, USA). The template plasmids pENT12-SURFIN_4.1^2Myc-N-T-Cyt and pENT12-SURFIN_4.1^{2Myc-N-T-Cyt-StuI} were generated using the Q5^® Site-Directed Mutagenesis Kit (NEB) [22 (link)] based on the initially generated pENT12-SURFIN_4.1^N-T-Cyt plasmids (Primer list; Additional file 1) [18 (link)]. For P. falciparum transfection, the region encoding WRD2 of SURFIN_4.2 was amplified by PCR with primers listed in Additional file 1. PCR fragments were analysed by electrophoresis on a 1.2% agarose gels, and purified by using TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 (Takara, Japan). The purified PCR product was ligated into the StuI site of pENT12-SURFIN_4.1^{2Myc-N-T-Cyt-StuI} plasmid to generate the pENT12-SURFIN_4.1^{2Myc-N-T-Cyt-4.2WRD2} plasmid. All constructs were verified by restriction digestion and sequencing. Ultimately, pENT12-SURFIN_4.1^2Myc-N-T-Cyt and pENT12-SURFIN_4.1^{2Myc-N-T-Cyt-4.2WRD2} plasmids were recombined with the destination plasmid pCHD43-II (modified based on pCHD-3/4 plasmid [23 (link)]) with pENT41-pfHsp86-5′UTR and pENT23-GFP_m2 using the Gateway Multisite LR recombination reaction according to the manufacturer’s instruction.

Three pairs of primers (Table 1) were designed to amplify the GPV Rep1 and GPV VP1, respectively. The PCR products were analyzed by electrophoresis in a 1% agarose gel. The DNA fragments from PCR were extracted by using the TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 (Takara, Dalian, China). According to the sequence of GPV YZ99–6, we designed three pairs of primers (Table 1) to amplify the right and left ITR. As expected, the ITR of GPV RC16 shown highly identity with the GPV YZ99–6. The fragments were cloned into the pMD19-T (Takara, Dalian, China) by TA clone and named pMD-GPV 1–187, pMD-GPV 188–412, pMD-GPV 412–2492, pMD-GPV 2493–4015, pMD-GPV 4016–4863 and pMD-GPV 4864–5046, respectively, which were directly sequenced at TSINGKE Biological Technology (Chengdu, Sichuan, China).
Table 1

The oligonucleotide primer used for amplification of GPV RC16 genome in this study

Primer	The sequence (5′-3′)
A1F	TCATTGGAGGGTTCGTTCGTTC
A1R	CATGCGCGCGGTCAACCTAACAGCCG
A2F	CGCGCGGTCAGCCCAATAGTTAAGCC
A2R	CTTCCTGGCGCGCAAAATATC
A3F	GATATTTTGCGCGCCAGGAAG
A3R	GAGGGGCTCCAGCTTTCAGATTCC
A4F	CGGAATCTGAAAGCTGGAGC
A4R	CTAAAATATTTTGGGCTGGGATGC
A5F	TCAGCTACTCACACAGAAG
A5R	CATGCGCGCGGTCAGCCCAATAG
A6F	CGCGCGGTCAACCTAACAGCCGG
A6R(A1F)	TCATTGGAGGGTTCGTTCGTTC