Controls are a critical part of a gene silencing experiment. They enable accurate interpretation of knockdown data and provide confidence in the specificity of the response. Changes in the mRNA or protein levels in cells treated with negative or non-silencing controls reflect non-specific responses in cells and can be used as a baseline against which specific knockdown can be measured.
Available controls for the TRC library include:
(1) pLKO.1 empty lentiviral vector: The pLKO.1 lentivirus plasmid vector does not contain an shRNA insert and is useful as a negative control in experiments using the TRC shRNA library constructs.
(2) eGFP shRNA-containing positive control: This control targets the eGFP reporter and has been validated to produce knockdown of eGFP in transduction-based assays at all multiplicity of infections (MOIs) tested.
Self-inactivating replication incompetent viral particles can be produced in packaging cells (HEK293T) by co-transfection of the pLKO.1 vector with compatible second or third generation packaging plasmids.
Each vial of the pLKO.1 control vector is shipped at a concentration of 0.5µg/µl in a total volume of 20µl, thus providing a total amount of 10µg vector DNA. The vector DNA is shipped in a microfuge tube at room temperature and should be stored at -20°C or -80°C.
