Quanti it picogreen dsdna reagent

Genomic DNA was extracted in triplicate from the soil samples using the MP FastDNA™ SPIN Kit for soil (MP Biomedicals, NSW Australia), as described (van Dorst et al., 2014 (link)). The DNA extracts were quantified using Quanti-iT™ Picogreen® dsDNA reagent (Invitrogen, Paisley UK) on a black 96 well plate at an absorbance of 520 nm using a fluorescence plate reader (SpectraMax M3 Multi-Mode Microplate Reader; Molecular Devices, CA). The DNA lysate concentrations produced ranged from 0.21 to 0.37 ng μl⁻¹ and were stored at −80°C until used further.

Lactoferrin and lactoferricin, generation 3-diaminobutyric polypropylenimine dendrimer (DAB) and the other chemicals were purchased from Sigma Aldrich (Poole, UK). The expression plasmids encoding Tumor necrosis factor (TNF) α (pORF9-mTNFα) and β-galactosidase (pCMVsport β-galactosidase) were obtained respectively from InvivoGen (San Diego, CA) and Invitrogen (Paisley, UK) and were purified using an Endotoxin-free Giga Plasmid Kit (Qiagen, Hilden, Germany). Passive lysis buffer was from Promega (Southampton, UK). Quanti-iT™ PicoGreen^® dsDNA reagent and tissue culture media were obtained from Invitrogen (Paisley, UK). Bioware^® B16-F10-luc-G5 mouse melanoma was obtained from Caliper Life Sciences (Hopkinton, MA). A431 human epidermoid carcinoma and T98G human glioblastoma were purchased from the European Collection of Cell Cultures (Salisbury, UK).

The prepGEM DNA extraction protocol was used as described in Ferrari et al. (2008 (link)) with some modifications. Firstly a quarter slice of each growth membrane was placed in a PCR tube with 99 μl of 1X Buffer (diluted from 10X) and 1 μl of prepGEM enzyme. Samples were mixed and placed in a thermocycler (Bioteke corporation), following the program of 37°C for 15 min, 75°C for 15 min, and 95°C for 15 min. The microfuge tube was centrifuged at 14,000 rpm for 3 min. The membrane slice was then removed with the help of sterile tweezers and 80 μl of DNA lysate was transferred to a new PCR tube. The genomic DNA was quantified using Quanti-iT™ Picogreen® dsDNA reagent (Invitrogen, Paisley UK) following manufacturer's instructions and stored at −20°C until used.