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Fees - Duke Cancer Institute Shared Resource

Below is a general description of our services and approved SOM FY 2018 fees.  Please contact Cheryl Bock for ES Cell and Embryo Services or Gary Kucera for Molecular Biology Services.  To download a request form click here.

 

Services and Fees for transgenic model generation via homologous recombination in ES cells:

Full service KO, KI cKO, cKI model via Homologous Recombination: $

We will generate a targeting vector (KO,KI, cKO, cKI) via BAC Recombineering techniques, prepare the vector for ES cell targeting, generate a MOCK control DNA and validated PCR protocol for screening targeted ES cells. Targeting vector will be transfected into ES cells.  Clones will be selected and PCR screened using validated PCR protocol.  The BAC core will prepare genomic DNA from up to 18 putative positive ES clones and validate these clones across homology arms via a combination of PCR and/or Southern blot. Positive ES cell clones that have been validated will be injected into blasts, and then transferred to pseudopregnant females for generation of chimeric offspring.  Chimeras will be passed to the PI for further characterization/matings, etc.

 

Target vector generation by BAC Recombineering:  $3717

We will generate a targeting vector (KO,KI, cKO, cKI) via BAC Recombineering techniques, prepare the vector for ES cell targeting, generate a MOCK control DNA and validated PCR protocol for screening targeted ES cells.

 

ES cell transfection with in-house generated targeting vector:  $3270

Targeting vector will be transfected into ES cells.  Clones will be selected and PCR screened using validated PCR protocol.  Up to 18 putative positive clones will be provided to the PI (or BAC core, see below) for further validation via Southern blot analysis.

 

ES cell screening of putative targeted clones (optional):  $1171

The BAC core will prepare genomic DNA from up to 18 putative positive ES clones and validate these clones across homology arms via a combination of PCR and/or Southern blot.

-DNA preparation only -  genomic DNA from up to 18 putative positive ES clones by BAC Core $500

 

Morulae microinjection of positive ES clones:  $3819

G4 ES cells from several positive clones confirmed by the contracting lab or by the DCI TG Core will be microinjected into ICR/Hsd morulae to produce chimeric mice. Blastocysts from C57BL/6J will be injected for 129 or B6 ES cell lines. This production method is less efficient.

 

ES cell microinjection with Repository clones provided by PI:  $2219

Repository clones provided by the PI lab (e.g. EUCOMM) will be injected into blasts (FVB/NHsd), and then transferred to pseudopregnant females for generation of chimeric offspring.  Chimeras will be passed to the PI for further characterization/matings, etc.

 

 

Services and Fees for transgenic model generation via CRISPR technology:

Full service CRISPR generated model (allelic analysis included):  $

We will generate up to three independent guide sequences and clone these into pX330 CRISPR vector. We will generate sgRNA from each of the three cloned guide sequences and screen for optimal activity using our in-vitro CRISPR screening protocol.  The sgRNA showing greatest activity will be used for pronuclear injections. The optimal sgRNA reagent and cas9 mRNA will be prepared and injected into the pronucleus of fertilized mouse embryos.  If no CRISPR effects (i.e. Indels) are identified (by CRISPR Founder screening), re-injections will be at no cost.  If indels are detected, but desired point mutations are not present (as determined by Allelic cloning(see below)), subsequent rounds of injection will incur additional charges at the original rate.  Genomic DNA will be prepared from tissue samples, followed by PCR amplification of the target region and Sanger sequencing. For further characterization of the indels, we will subclone the individual alleles from up to 6 founders.  These clones will be sequenced to identify the specific modifications to the target locus. 

 

CRISPR guide RNA design and cloning:  $2652

We will generate up to three independent guide sequences and clone these into pX330 CRISPR vector.

 

CRISPR sgRNA Production via In-vitro Transcription:  $1172

We will generate sgRNA from each of the three cloned guide sequences and screen for optimal activity using our in-vitro CRISPR screening protocol.  The sgRNA showing greatest activity will be used for pronuclear injections.

 

Pronuclear Injection of CRISPR Reagents:  $2816

The optimal sgRNA reagent and cas9 mRNA will be prepared and injected into the pronucleus of fertilized mouse embryos.  If no CRISPR effects (i.e. Indels) are identified (by CRISPR Founder screening,) re-injections will be at no cost.  If indels are detected, but desired point mutations are not present (as determined by Allelic cloning(see below)), subsequent rounds of injection will incur additional charges at the original rate.

 

Founder Analysis, Initial:  $921

Genomic DNA will be prepared from tissue samples, followed by PCR amplification of the target region and Sanger sequencing.

 

Founder Analysis, Advanced, Allelic Subcloning:  $1765

Note:  This service is required for requests involving base pair changes.

 For further characterization of the indels, we will subclone the individual alleles from up to 6 founders.  These clones will be sequenced to identify the specific modifications to the target locus.

 

Services and Fees for transgenic model generation via BAC Transgene:

BAC modifcations for Transgenics:  $3197

We will eliminate the single loxP site within the backbone vector of the BAC clone upon request.  We will modify the BAC clone per request (may include insertion of an ORF, reporter gene, etc).  BAC DNA will be maxi prepared and linearized for pronuclear injection.

 

Pronuclear Injection:  $2816

Linearized BAC DNA will be prepared and injected into the pronucleus of fertilized mouse embryos.   Additional injections, if required, will incur new charges at the original rate.