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Lentivirus Production
Published: Mar 5, 2011 DOI: 10.21769/BioProtoc.39 Views: 51490
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Abstract
Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid.
Materials and Reagents
- 293T packaging cells (ATCC)
- Hairpin-pLKO.1 vector (GE Healthcare Dharmacon)
- Packaging vectors: 2nd generation packaging plasmids containing gag, pol, and rev genes (pCMV-dR8.91 or pCMV-dR8.74psPAX2) and envelop plasmid (pMD2.G) (Addgene)
- Lipofectamine 2000 (Life Technologies, Invitrogen™)
- OptiMEM serum free media (Life Technologies, Invitrogen™)
- DMEM (Life Technologies, Invitrogen™)
- Fetal Bovine Serum (FBS) (Life Technologies, Invitrogen™)
- Pen/Strep (Life Technologies, Invitrogen™)
- 6 cm cell culture plates (Thermo Fisher Scientific)
- 45 μm filter (Thermo Fisher Scientific)
- Storage tubes
- Seeding media (see Recipes)
- Harvest media (see Recipes)
Equipment
- Tissue culture incubator (37 °C, 5% CO2)
- Centrifuges
Procedure
Category
Microbiology > Microbial biochemistry > RNA
Cell Biology > Cell isolation and culture > Cell growth
Molecular Biology > RNA > Transfection
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