...HIV-derivative viruses. For transient knockdown of protein expression, you may transfect plasmid DNA directly...post-infection mRNA knockdown (quantitative PCR) ≥3 days Protein knockdown (western blot) ≥4 days Phenotypic assay...plate so as not to disturb the transfected cells. k. Incubate cells at 37°C, 5% CO 2 for 24 hours. Day...Vector Element U6 Human U6 promoter drives RNA Polymerase III transcription for generation of shRNA transcripts...because polyT is a termination signal for RNA polymerase III. Note that these were the prevailing guidelines...to adjust the sequencing conditions if the DNA polymerase has difficulty reading through the secondary ...