Ordering Custom Constructs

Vaccinia Description and Custom Construct Information-

Vaccinia virus is a member of the Orthopoxivirus genus. Recombinant vaccinia is a very useful vector because of its stability, ability to obtain high titer stocks, and its capacity to incorporate up to 70 kb of foreign DNA. Protein is expressed rapidly and at a high level. Vaccinia shows broad tropism in vivo and in vitro.

The recombinant vaccinia virus is produced by homologous recombination between a plasmid and the viral DNA within infected cells. The gene of interest is inserted in a non-essential locus of the vaccinia DNA. To aid in the recovery of the recombinant virus, plasmids have been generated that allow for selection of the recombinant vector.

Since vaccinia encodes its own DNA-dependant RNA polymerase, which does not recognize foreign promoters, the cDNA must be cloned downstream of a well defined virus promoter or close to an endogenous vaccinia promoter.

Vaccinia viruses are widely used for the study of poxvirus molecular biology, functional characterization and over expression of proteins, and as candidates for live vaccines or vaccine research.

How to Order:

  1. Have your sequence, maps, digest, and gene information prepared. It is imperative that we follow NIH guidelines for recombinant DNA technology.  Therefore, all work must be approved by our Recombinant DNA Advisory Committee. Complete gene information must be completed on the Service Request Form. University of Iowa Investigators need a copy of the approved rDNA form.
  2. Ordering is placed online at through iLabs Solutions. Please click the link at the top of the page.
  3. Register for an iLabs Account.
  4. Log into the iLabs Website
  5. Select the New Constructs and Miscellaneous Services only bar under the Request Services Tab.
  6. Select: Vaccinia Virus (New Constructs and Miscellaneous Services only).

Services:

Amplification, purification and titer from viral particles:

  • Sample required: Viral Particles
  • Timeline: 3-4 weeks from the time the virus is received
  • Service:
    1. Large-scale amplification.
    2. Purification by sucrose cushion
    3. With every new lot an infectious titer (pfu/ml) is provided.
  • Material provided: 8ml of purified virus at a concentration ranging from 1 x10^7 pfu/ml to 1 x10^9 pfu/ml.
  • Vehicle: Vaccinia vectors are re-suspended in 10mM Tris HCl pH 9.0.
  • Cost: $2,600.00 per construct plus shipping and handling.

Construction, selection, purification, amplification and titer:

  • Shuttle: You need to purchase your own packaging/ expression plasmid.
  • Sample required: 20 µg of vaccinia shuttle plasmid expressing the gene of interest at a concentration greater than 0.25 µg/µl.
  • Timeline: 6-8 week from the time the plasmid is received
  • Services:
    1. Transfection 
    2. Three rounds of selection
    3. Large-scale amplification
    4. Purification by sucrose cushion
    5. With every new lot an infectious titer (pfu/ml) is provided
  • Material provided: 8ml of purified virus at a concentration ranging from 1 x10^7 pfu/ml to 1 x10^9 pfu/ml.
  • Vehicle: Vaccinia vectors are re-suspended in 10mM Tris HCl pH9.0.
  • Cost: $3,000.00 per construct plus shipping and handling.