Plasmid dna mini kit 1

The pCMT-flp plasmid, which confers Tet resistance, was purified from a common cloning strain of E. coli, E.cloni 10G (Lucigen), using E.Z.N.A. Plasmid DNA Mini Kit I (Omega Bio-Tek) following manufacturer's protocol, and was eluted at a final concentration of 11 ng/μl. Electrocompetent E.coli SM10 λpir were prepared as described above. pCMT-flp plasmid DNA (11 ng) was introduced into electrocompetent E. coli SM10 λpir via electroporation following the protocol above, but with a single pulse of 1.6 kV, 25 μF, and 200 Ω, and incubated at 37°C with shaking at 200 rpm for 4 h. Cells were then plated onto 2xYT+10 μg/ml Tet. Presence of the pCMT-flp plasmid was verified by the ability of colonies to grow on Tet-selective media.

The pGEM-T vector containing EBNA1 was transformed into a competent cell (E. coli, DH5α). Bacteria carrying the plasmid were grown and extracted for the plasmids using E.Z.N.A.^® Plasmid DNA Mini Kit I (E.Z.N.A.^® Plasmid Mini Kit I, Omega Bio-tek^®, Norcross, GA, USA) according to the manufacturer’s instructions. The concentration of plasmid was quantified by NanoDrop. The presence of EBNA1 was detected by PCR using EBNA1 primer pair (Table 2) and constructed the standard linear regression equation.
To determine whether andrographolide inhibits EBV virion production, EBV copy number was determined in the supernatant collected from cells that were either treated with CC₁₅ or CC₂₅ of andrographolide or NaB alone or a combination of CC₁₅ or CC₂₅ of andrographolide and NaB for 48 h. The supernatant was harvested by centrifugation and further extracted for DNA by adding cell lysis solution according to the manufacturer’s instructions (QIAGEN, Redwood City, CA, USA). The copy number of EBV genomes was estimated from the standard curve of EBV copy number by using CT values obtained from samples and the standard linear regression equation. The results were expressed by means of copies/50 ng of DNA derived from three independent experiments.