We clone everything:
Epitope tags: N-, C-terminal, and between the protein domains
Mice knock-out, knock-in, and transgenic constructs
Regular cDNA cloning starts from $150.00 depending on the size of the insert. Price includes primer design and synthesis, sequencing verification of 5' and 3' ends of insert. Deliverables are one mini-prep, one glycerol stock and electronic report with sequencing chromatograms.
Using BAC recombineering, we can generate constructs in as little as 6 weeks from initial consultation to final sequencing verification. By performing restriction analyses verification at key steps of the process, we have increased the quality and reliability, as well as the speed, of vector construction. We can create BAC transgenic constructs for uncharacterized promoters. This technique allows insertion of large (>20 kb) genomic sequences from BACs into smaller vectors suitable for gene targeting. This process bypasses the restraints of conventional ligations to produce longer genomic “arms” to increase the efficiency of homologous recombination in ES cells. Moreover, PCR error in these arms is circumvented since genomic DNA in the BAC clones is manipulated to generate the DNA constructs. Site-directed mutagenesis, point mutations, or any kind of Knock-In constructs pose no problems for this technology. We have the capacity and flexibility to tailor-make elements for targeting and transgenic constructs (including BAC transgenic). Additionally, we are currently working at implementing a yeast system to perform many steps even more rapidly and reliably.
To get started, we need a gene name and mutant model type – i.e. KO, KI, transgenic, etc. We will put together a design and send for your approval. Before ordering primers, we require payment in the form of Purchase Order, Credit Card, Wire transfer, or check agreement (please contact email@example.com for additional billing details)
We provide everything else.
Here is a summary of what we provide per construct:
Maps/sequences of final construct, all intermediate vectors, mutant locus, etc.
Design for model
Final plasmid sent on filter paper
Raw sequencing data of final construct
Upon request, we can also provide (at no additional cost):
Methods section-we suggest acknowledgment or authorship on publication
Glycerol stock of final plasmid (if courier account number is provided)
Glycerol stock of parent BAC clone (if courier account number is provided)
Gel images of:
PCR verification steps
Restriction Verification steps
For final verification of targeting constructs, we perform 5 verification digests and sequencing over all insert junctions (6 junctions total for conditional KO).
For targeting constructs, the final construct is in a high-copy plasmid, so there is no need to culture BAC clones and extract DNA from BAC clones.
For BAC constructs, we provide sequencing verification across all insert junctions.
Library of available cassettes:
Cre-ERT2 (Tamoxifen-Inducible Cre)
Tet-On (aka rtTA [reverse transcriptase transactivator])
TRE2 (aka Tet-Responsive Element2, or “tet-O-”)
Conditional Gene-trap cassette
Floxed STOP cassettes
We can custom design these to include an upstream IRES, loxP sites, frt sites, rox sites, bacterial selection cassettes, mammalian selection cassettes, etc.
Cloning & Sub-cloning Service
Constructs for Adenovirus Expression System
It is a very strong and efficient system for the simultaneous expression of two or more specific gene(s) in mammalian cells. We provide this service for the construction of your gene of interest. Toxin-free final product is ready to transfect HEK cells for the production of recombinant adenovirus.
Constructs for Baculovirus Expression System
One of the most widely used gene(s) expression system for production of recombinant proteins in Eukaryotic cells (such as sf9, Sf21). We offer cloning services for the construction of gene of your interest in baculovirus-based expression vector(s). Final product is ready to transfect insect cells for the packaging of recombinant baculovirus.
Construction of Gene-specific SiRNA
This revolutionary technique to block the expression of a specific protein(s) in cells or animals has proven to be a reliable and quick approach. We'll take care of your SiRNA construct from designing of the specific primers to its cloning in an SiRNA expression vector. We also provide SiRNA duplex synthesis service with desired modifications such as biotin.
This service is provided for sub-cloning of gene/cDNA into vector of your choice. For accuracy, recombinant clones will be verified by restriction analysis. A project report with detail analysis and 10 ug of final product will be supplied. Customer supplies the specific gene/ cDNA and vector.
We'll design and make gene-specific primers for PCR amplification from a cDNA library. We'll buy commercially available specific cDNA library if customer can not provide it. PCR amplified cDNA fragment will be sub-cloned in TA cloning vector or in a vector of your choice. Recombinant clones will be verified by restriction fragment analysis. For accuracy, two selected clones will be sequenced. A project report with detail analysis and 10 ug of final product will be supplied.
We'll reverse transcribe and PCR amplify specific gene from customer’s provided total RNA/mRNAs. Amplified fragment will be ligated in TA vector or in vector of your choice. Two clones will be sequenced for the validity of clones. A project report with detail analysis and 10 ug of final product will be supplied.
cDNA Library Construction
We offer cDNA library construction service at very competitive price.
We charge $295/plasmid this includes cloning of a single insert and addition of ~20 bases of new sequence (no template required). Addition of longer sequences cost $10 per 20 bases. Cloning of Inserts longer than 4kb or cloning of more than one insert may result in additional charges, please request a quote. Sequencing charges are $15 per 800 bp (covers primers and sequencing costs)*.
Our service: Sub-clone DNA into plasmids according to customer's request.
Sub-clone your gene to another plasmid, make any sequence modification to your gene or plasmid. Our procedures allow cloning not only by using restriction enzymes but also in restriction-enzymes-independent ways. This allows sequence modification in areas where no suitable restriction sites are present.
Examples for services offered:
Amplification of insert from one plasmid and cloning in another plasmid.
Addition of sequence to an existing plasmid, for example adding FLAG+myc tags, restriction sites, linkers, shRNA coding sequences.
Introducing site directed mutations.
Deletion of sequences of any length from a plasmid.
Insertion of one gene fragment in to another gene, for example for making a chimeric protein.
Cloning an insert in to a plasmid while adding a tag at the 5' or 3' ends of the insert.
Cloning an insert while adding a polyglycine spacer to prevent structural effects of tags on your protein.
Any other sequence modifications you request.
* Please request a specific quote in the following cases:
Cloning into viral vectors.
Cloning of repeated sequences.
Cloning of AT- or GC-rich sequences.
Cloning of Inserts longer than 3.5 kb.
Site directed mutagenesis in plasmids larger than 9 kb.
Cloning to vectors with no full sequence information.
Cloning of more than one insert.
How it works:
Send us description of your source DNA and how you want it cloned.
Mail us your source plasmid/s.
How to ship samples:
Please provide 5 micrograms of the source plasmid/s as purified DNA good for sequencing at concentration ≥0.2 mg/ml. Miniprep DNA is fine. Please use overnight carrier with tracking service i.e. FedEx, UPS, DHL and ship at ambient temperature. Forward us the tracking number if possible.
We send you the completed plasmid and sequencing information in 2 - 4 weeks.
PCR based cloning $0.25/bp (Minimum 1kb gene)
Clone library creation:
In order to confirm bacterial identity, PCR-amplified 16S rDNA fragments can be cloned into plasmids and transformed into an appropriate host (usually E. coli). Each transformed E. coli colony will contain only one 16S rDNA PCR-originated fragment from the original sample. A number of colonies are picked randomly and the insert is sequenced. The DNA sequence is then compared to a database (for example RDP II) for identification by DNA homology.
Genetix QPix2 XT Colony Picker System
UNC researcher = $110/sample
Non-UNC researcher = $162/sample
construction of transgenic or knock out insertion vector
Cloning of DNA fragments into the vector of choice
Turnaround time: 3 days
ViGene offers a broad range of custom cloning services at a reasonable cost and speed delivery. Our highly experienced scientists are ready to tackle any of your difficult cloning projects, including unstable clones, very large clones, toxic clones, and clones with high GC content and highly repetitive sequences. Just send your sequence or clone, we will do the work for you.
●Subclone the ORF from our Entry clone to any of our destination vectors with different fluorescent or affinity tags at either N-terminal or C-terminal
●Synthesize or clone any DNA sequence from any organisms to any of our vectors
●Subclone to our adenoviral or lentiviral vectors
●Clone any DNA sequences or our full-length ORF into your own vectors
●All clones will be fully sequenced to match your specifications
●Deliver 10ug DNA or glycerol stock as requested
●Subcloning cost is $200 each
We also offer a world class adenovirus collection.
• Choose from more than 1,200 human adenoviral miRNA clones or/and more than 1,200 packaged, tittered & ready-to-use miRNA adenovirus
• Choose from more than 15,000 human adenoviral ORF cDNA clones or/and premade ORF adenovirus