pLVTHM
(Plasmid #12247)

• Purpose: (Empty Backbone) 2nd gen lentivector expressing shRNA from H1 promoter and GFP. For direct cloning of shRNA or sub-cloning of an H1-shRNA cassette from pSUPER. Dox-controllable if used with tTRKRAB expressing plasmid
• Depositing Lab: Didier Trono
• Publication: Wiznerowicz et al J Virol. 2003 Aug . 77(16):8957-61.

Backbone

• Vector backbone: pLVTH
• Backbone size (bp): 11087
• Vector type: Mammalian Expression, Lentiviral, RNAi, Cre/Lox

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Growth instructions: Use Stbl3 or HB101 to reduce chance of recombination. Grow at 37C
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: None

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: See map

Resource Information

• Addgene Notes:
  • Addgene Diagnostic Digest
• Articles Citing this Plasmid:
  • 180 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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.

Information for Cloning Grade DNA (Catalog # 12247-DNA.cg) ( Back to top)

Purpose

Cloning grade DNA is suitable for use in PCR, cloning reactions, or transformation into E. coli. The purity and amount is not suitable for direct transfections.

Delivery

• Amount: 2 µg
• Guaranteed Concentration: 100 ng/µl +/- 5 ng/µl
• Pricing: $105 USD
• Storage: DNA can be stored at 4℃ (short term) or -20℃ (long term).

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Quality Control

Addgene has verified this plasmid using Next Generation Sequencing. Results are available here