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What techniques are critical to successful packaging with Trans-Lentiviral packaging system?

A few of the more critical techniques are listed below: 1. Make sure the HEK293T cells are not split too thin. In other words, do not split the cells more than 1: 5. And in the days leading up to transfection, split or expand the cells 1: 2 so that the cells are in a "rapid" replication state. Do not split the cells back so far that it takes 5 days to become confluent. It just slows down the cellular machinery to a crawl. You want the cell's transcriptional and translational machinery functioning at their highest level when generating virus. 2. Transfect the cells within 24 hours after seeding and at a confluency of no less than 90%. This is why you need the cells rapidly replicating. 3. The transfer vector DNA (i. e. SMARTvector, pGIPZ, pTRIPZ) should be of transfection quality. If using Qiagen, we prefer maxiprep DNA over midiprep or miniprep.