One Shot™ Stbl3™ Chemically Competent E. coli

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.

We strongly recommend using Stbl3 E.coli for cloning lentiviral constructs. Stbl3 E.coli contain the recA13 mutation in their genotype that helps to minimize the likelihood of unwanted recombination between the LTRs. After transforming into Stbl3 E.coli, we recommend picking colonies and validating the Lenti DNA from mini-preps, using Afl II and Xho I digests before proceeding to midi-preps. In all of our lentiviral vectors, Afl II sites are present in both 5´ and 3´ LTRs, and an Xho I site is present after the 3´ end of the MCS. Assuming Afl II cuts only in the LTR sites, and there are no Afl II or Xho I sites in the insert, 3 DNA fragments are expected to be generated from the Afl II + Xho I digest. Any unexpected DNA fragments can be assumed to be a result of LTR recombination. Only clones with the expected pattern of DNA fragments should be chosen for the subsequent midi-prep.