...from an AgeI/EcoRI restriction digest) David Root 10879 pLKO.1 ‐ TRC control Negative control vector containing...Retroviral vector for shRNA expression Bob Weinberg 10675 pMKO.1 puro GFP shRNA Negative control vector for ...protocol for using the popular cloning vector pLKO.1. Trono Lab Lentivectors Toolbox Tyler Jacks Lab Protocols... Lentiviral vectors for constitutive shRNA expression ID Plasmid Description PI 10878 pLKO.1 ‐ TRC cloning...non-hairpin insert David Root 1864 pLKO.1 ‐ scrambled shRNA Negative control vector containing scrambled shRNA...shRNA cassette from another vector (e.g. pSUPER) can be cloned into this vector; Contains a 5'LTR and should...Retroviral vectors for constitutive shRNA expression ID Plasmid Description PI 8452 pMKO.1 puro Retroviral...

...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...