Pgem t easy vector system 2

The amplified fragments (ASFV-QP509L, ASFV-Q706L, ASFV-CP204L, ASFV-B646L, and Cyclophilin A) were cloned into a plasmid vector (pGEM-Teasy Vector System II, Promega, Madison, USA), and used to transform DH5α competent cells. Then, plasmids were isolated from bacteria using the Roche High Pure Plasmid Isolation Kit (Roche Applied Science, Germany), according to the manufacturer’s manual. To determine whether the cloned DNA fragments were incorporated into vectors, the inserts were amplified by PCR and their sequences were confirmed. Following this step, the concentration of each plasmid preparation was determined by spectrophotometric absorbance (NanoDrop 2000c). Their corresponding copy number was calculated using the equation: pmol (dsDNA) = μg (dsDNA) × 1515 / DNA length in pb (pmol = picomoles, dsDNA = double-strand DNA, DNA length in pb = number of base pairs from the amplified fragment; 1 mol = 6.022 × 10²³ molecules).

DNA methylation was analyzed by bisulfite conversion (EpiTect Bisulfite Kit, Qiagen) of genomic DNA from differentiated myocytes. To perform the bisulfite analysis of the 4qA-L allele, previously published [21 (link)] PCR oligonucleotide primers (BSS4qALF for forward 5′-TTATTTATGAAGGGGTGGAGTTTGTT and BSS3626R for reverse 5′-AACAAAAATATACTTTTAACCRCCAAAAA) were utilized. All PCRs were performed using the hotstart Taq (JumpStart REDTaq from Sigma) at 95 °C for 5 min, 95 °C for 30 s, 55 °C for 30 s, 40 cycles at 72 °C for 1 min and 72 °C for 1 min. The amplified 354-bp PCR product was then gel purified and cloned into pGEM-T-Easy vector system II (Promega) and transformed into DH10B electrocompetent cells. The DH10B cells that carried the ligated vectors were selected by color change on agar plates containing the ampicillin/X-gal/IPTG. The white colonies represented the vectors with the PCR product, while the blue colonies represented empty vectors. The white colonies were picked and bulked in LB medium. The plasmids containing the target DNA were extracted by using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced to reveal methylation distribution at a single molecule level.

Total RNA was extracted from 150 mg flash-frozen individuals of the fire ant (whole body) using the TRIzol kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using a PrimeScript kit (Takara Biotechnology, Dalian, China). PCR thermal cycling was performed as follows: an initial denaturation at 94°C for 3 min followed by 35 cycles at 94°C for 30 s, 60–45°C (depending on the primer pair) for 30 s, and 72°C for 1–3 min (determined by the length of the amplified fragment) with an additional polymerization step at 72°C for 10 min. The PCR products were cloned and sequenced using the pGEM-T Easy Vector System II (Promega, Madison, WI).