One Shot™ Stbl3™ 化学感受态大肠杆菌

我们推荐使用Stbl3感受态细胞，因为它们已经经过测试可以用于克隆含有直接重复序列的不稳定慢病毒DNA。

We would recommend our Stbl3 competent cells, as they have been tested for cloning of unstable lentiviral DNA sequences containing direct repeats.

• Use low passage 293FT cells. Do not use 293FT cells beyond passage 20. Freeze down many aliquots and grow for 2–4 passages prior to transfection.
• Passage cells in complete D-MEM containing G418 (500 µg/mL). Supplement the media with "non-essential" amino acids and sodium pyruvate (0.1 mM MEM Non-Essential amino acids and 1 mM MEM Sodium Pyruvate). Use Gibco FBS (Cat. No. 16000-044).
• Plate cells at a density of 5 x 10e6 per 100 mm dish. Cell density is very important. Make sure that the cells are growing well before re-plating prior to the day of transfection. Avoid overgrowth of 293FT cells when passaging.
• When plating for transfection the next day, do not add G418 to the media.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

We do not recommend using mini-prep plasmid DNA for lentivirus production. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K1910-01) or PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004) which contain 10 mM EDTA in the Resuspension Buffer. Since lenti DNA midi-preps also often have low DNA yields, we recommend following specific protocols to increase yield—basically, grow cells slowly, use fewer cells per column, and use 100 mL lenti culture for each DNA midi-prep.

Our ViraPower lentiviral expression system is a 3rd generation system with regard to safety features. Our lentiviral expression vectors are derived from wild type HIV, but nearly all the wild type viral proteins (e.g., Vpr, Vpu, Vif, Nef, Tat) have been removed and the HIV envelope is not used. VSV-G (vesicular stomatitis virus G) envelope protein is used instead. Our ViraPower lentiviral expression system can be used with a 2nd generation lentiviral packaging mix. However, our lentiviral packaging mix would not be compatible with a 2nd generation lentiviral expression vector.