Lipofectamine® 2000 Transfection Reagent

Protocol tips

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	Do not add antibiotics to the Transfection Medium

Protocol tips
Do not add antibiotics to the Transfection Medium

Publication protocol

The ratios of transfection reagent to DNA examined are listed in Table S1 (Supplementary material). For Linear PEI 25,000 Da (Polysciences Europe GmbH, Germany) the protocol is as follows (based on [8]): 0.8 mL of DNA and varying amounts of PEI (Table S1) were diluted in separate tubes containing 8.31 mL of 150 mM NaCl, mixed and incubated at room temperature for 2 min prior to addition to the cell suspension in CD-CHO medium (16.62 mL of complex in 500 ml of cells). For Lipofectamine® 2000 (Invitrogen, UK), the DNA and various amounts of Lipofectamine® 2000 (Table S1) were each diluted separately in 50 mL of Opti-MEM® I Reduced Serum Medium (Invitrogen, UK), mixed and incubated at room temperature for 5 min. The diluted DNA and Lipofectamine® 2000 were combined and incubated at room temperature for 20 min before addition to the cells. For the TransIT-PRO® transfection, TransIT-PRO® and PRO Boost reagents (both Mirus Bio LLC, US) were pre-warmed to room temperature. The TransIT-PRO®, DNA and PRO Boost complex was formed by adding appropriate amounts of each reagent into DNA that was diluted in 100 mL CD-CHO medium (Table S1). Complexes were incubated at room temperature for 30 min prior to addition to the cell suspension. In all cases triplicate samples and negative controls were examined. Transfection efficiency by each reagent was determined via the fluorescent emission of EGFP protein expressed by the cells upon DNA uptake. EGFP has a maximum excitation wavelength of 488 nm (filter bandwidth 465 +/− 35 nm) and a maximum emission of 507 nm (filter bandwidth 510 +/− 10 nm). Fluorescent intensity was detected by the Infinite 200Pro microplate reader (Tecan Group Ltd, UK) every 2 h for 14 days, with a gain value of 100.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Lipofectamine® 2000 Transfection Reagent below.

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