...double digests. Digest pLKO.1 TRC-cloning vector with AgeI. Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep... ddH 2 O. Digest eluate with EcoRI. Mix: 30 μL pLKO.1 TRC-cloning vector digested with AgeI 5 μL 10x NEB...released by digestion with AgeI and EcoRI. shRNA oligos are cloned into the AgeI and EcoRI sites in place...use of this vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the...into pLKO.1 The pLKO.1-TRC cloning vector contains a 1.9kb stuffer that is released upon digestion with...ends of EcoRI and AgeI. Forward and reverse oligos are annealed and ligated into the pLKO.1 vector, producing...restriction digest with EcoRI and NcoI: 1 μg miniprep DNA 2 μL 10x NEB buffer for EcoRI 0.8 μL EcoRI 0.8 μL...