CLONING SERVICE
Duke Viral Vector Core is pleased to offer an expanded and cost effective Molecular Cloning services. We would be happy to help with custom gene cloning and vector construction, shRNA, CRISPR/Cas9 synthesis, plasmid DNA and RNA purification, and other projects.
We have proven expertise in designing and producing complex DNA constructs for nearly any purpose.
Custom Gene Cloning and Vector Construction
- Cloning of cDNA, ORFs from any species in the lentiviral and AAV vector backbones
- Protein-expression vectors for bacterial, and mammalian cell expression
- We supply sequence-confirmed plasmid DNA and plasmid maps
- Cloning using restriction enzyme digestion: sticky-end, blunt-end cloning
- Cloning using PCR amplification: TA cloning or blunt-end cloning.
- Cloning by Gene Synthesis: we work with The GeneArt™ Gene Synthesis service
http://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis.html to synthesize an DNA fragments and oligos.
- Cloning by Gibson Assembly: to assemble several DNA fragments in correct order for gene synthesis, synthetic biology, and novel expression vectors.
- Cloning by RT-PCR: Isolated RNA/mRNA from any tissue is reverse transcribed and cloned into a plasmid-of-interest.
- Viral Expression Vectors: the core has an extensive collection of in-house AAV, Lentiviral and Retroviral vector backbones, with combination of heterologous envelopes, genes, promoters and tags.
- Gene Targeting Vectors: Vectors for homologous recombination, knock-in, knock-out, Cre, FLP, and Flox constructs for genome integration.
- Inducible Expression Vectors: Conditional and inducible systems for a temporal and spatial controlled activation of genes including Tet-On, Tet-Off, and tamoxifen-inducible.
- Cloning of miRNA, mi-shRNA and shRNA into a plasmid-of-interest- pol II or pol III-promoter systems are available for transient and stable expression.
Mutagenesis: Additions, deletions, substitutions, site-specific and random mutagenesis.
We can help you with your cloning strategy, whether we start from your ready-to-use insert or whether we help you to customize your vector construction.
Quality control: double-strand sequencing of full insert. The end-point plasmids released to investigator/used for vector production after we verify their sequence by digestion with restriction enzyme and Sanger sequencing. The integrity of inverted-terminal repeats (ITRs) of AAV-plasmids is routinely verified by digestion with restriction enzymes prior to vector production.
