URL: http://www.addgene.org/12247

Proper Citation: RRID:Addgene_12247

Bacterial Resistance: Ampicillin

Defining Citation: PMID:12885912

Vector Backbone Description: Backbone Size:11087; Vector Backbone:pLVTH; Vector Types:Mammalian Expression, Lentiviral, RNAi, Cre/Lox; Bacterial Resistance:Ampicillin

Comments: pLVTHM allows for direct cloning of shRNAs into the lentiviral vectors and replaces the old pLVTH system from Trono lab. pLVTHM is similar to pLVTH, but contains a 3bp substitution that generates a unique MluI site for direct cloning of an shRNA into MluI-ClaI. Please note that there two additional Dam-methylase-sensitive ClaI sites in this vector that are blocked by Dam methylation (this does not appear in the depositor's full sequence) around the EF1a promoter. The plasmid needs to be grown in a dam(-) bacterial strain in order to use ClaI for cloning this region. Otherwise, dam(+) strains can be used for MluI-ClaI cloning shRNA. Note that ClaI has lower salt concentration requirements than MluI. One way to handle this is to digest with ClaI for 45 minutes, followed by addition of diluted MluI buffer and MluI, then incubation for 90 more minutes. Be sure not to use a large amount of vector (1.7 ug to 2.5 ug is known to work). Also, if your shRNA is already in pSUPER (or another plasmid under the control of the PolIII promoter) you may use the EcoRI-ClaI sites to replace the H1 promoter in pLVTHM with the H1-shRNA cassette from pSUPER (or another plasmid). The packaging plasmid for Trono lab lentiviral vectors is also available at Addgene http://www.addgene.org/rnaitools Please note that the full sequence for this plasmid is approximated and not fully verified. Please visit the Trono lab http://tronolab.epfl.ch for cloning strategies, protocols, publications, and more. See LentiWeb http://www.lentiweb.com for discussions on cloning strategies and protocols.

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