Why do you add one more C after BamHI site in the siRNA insert?
The transcription of pol III promoter starts at position 8. That extra C is to make sure that the siRNA insert gets transcribed correctly. • Why is my lentiviral virus titer from pRNA-U6.1/lenti or pRNATin-H1.2/lenti low? A: It is normal since the transfection efficiency and the copy number transfected is all different from cell to cell. Just make sure optimal ratio of target plasmid and packaging plasmids. The lentiviral titer is lower than other viral system. Invitrogen people suggest that transfection of 293FT packaging cell in suspension may improve the titer. I attached the protocol. And also you may transduce several times with lower titer stock every other day in order to accumulate cGFP positive cells. Make sure cells are not completely confluent in order to maintain active growing condition and during this series of transductions you may split cells.
