Titration of Lentiviral Vectors
Day 1 (9-12am)
- plate 30.000 293t per well on 24 well plate in 1 ml medium/well
Day 2 (4-6pm)
- count cells on 1 well (should have 60-80.000)
- transduce the cells with 6, 4-fold serial dilutions in 250µl total vol; take 50µl for non-conc. or 1µl for conc. vector stock for as a first dilution
Day 3 (9-12am)
- add 1ml of medium
Day 5
- split cells and analyze fluorescence by FACS and read percentage from linear values (usually 5-10%) Titer is a number (percentage) of cells transduced by a given vol and counted on D2 e.g. 1µl gives you 10% of positivie cells and you had 50.000 cells on D2, so you have 5.000TU/µl --> 5x106 TU/ml
Day 7-8
- isolate DNA for QPCR
© Maciej Wiznerowicz