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
Equipment
Procedure
Recipes
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Like for cells, FBS serves as a stabilizer during long term storage of virus. The bovine serum albumin in FBS preserves the virus by preventing ice Krystal formation. This reduces loss of virus infectivity after freeze and thaw cycles. Other stabilizers can be used, such as sucrose.
I have not tested that. So I don't have a good answer for that. But I think that few hours storage at RT may result in loss of the virus titer. The only way to find out to is check the titer. Adenovirus however are more stable at RT than lentivirus (up to 72 h). Hope that is helpful.
Lentivirus stock can be stored at 4C for for up to 5 days. No significant effects on the titer have been reported at those conditions.
This is a link to a paper where they study the factors affectinglentivirus titer.http://www.aaronlogan.com/downloads/Logan_infectivity.pdfClontech has a kit for lentivirus concentration that would increase the titer by 100 fold. Also ultracentrifugation can be used to concentarte lentivirus.I hope this answers the question.
It is recommended to use Harvest media that contains more FBS than Seeding media to provide the 293T packaging cells with additional nutrients while producing the virus particles. Also antibiotics (Pen/Strep) are added to the Harvest media to avoid bacterial infection of packaging cells. It is not recommended to use antibiotics in Seeding media while transfecting the cells using Lipofectamine 2000 as this causes cell death.