According to the growth characteristics of baculovirus, within a certain range, the inoculation titer of baculovirus is directly proportional to the production of target protein. Therefore, it is very important to determine the optimal titer of the virus. Creative BioMart offers two methods to determine the accurate recombinant virus stock titer—— quantitative PCR assay and plaque assay. Each of the two methods has its advantages. Customers can choose either one of them or a combo of the two method to obtain a more accurate result.
Creative BioMart has developed a rapid system based on quantitative PCR (qPCR) for the accurate titration of baculovirus stocks. In this method, the baculovirus DNA is purified from the budded virus using freshly amplified virus inoculum. The purified DNA is added to a qPCR reaction mix and then amplified in our Sequence Detection System. Following amplification, the quantity of virus particles is determined against the known standards that have been titrated by plaque assay.
Note. While this method has been proven to be as accurate as plaque assay in our experience, it is not recommended for virus stocks older than three months. Spurious results are obtained from old virus stocks as a result of aggregation and degradation of the virus particles. A plaque assay can determine the titer of an old virus stock more accurately.
Our service offers an optimized method for titration of virus, based on the plaque assay, to determine an accurate infectious titer of the recombinant baculovirus stock. Customers need to provide at least 0.5ml of virus inoculum for the qPCR titration method.
In the plaque assay, cell monolayers are infected with a serial dilution of the virus stock solution and an agarose overlay is used to restrict the flow of virus. Cells that are infected release progeny virus, however, this only allows infection of neighboring cells. Lysis of the infected cells produces clear regions or plaques. The plaques are then visualized using a dye such as neutral red.
This service offers an optimized method, based on the plaque assay, to determine an accurate infectious titer of the recombinant baculovirus stock.
The viral titer allows infection of the cells to be performed at a known multiplicity of infection (MOI). To achieve maximum production of recombinant protein, an optimal MOI is used. Amplification of virus stocks is also best achieved by using a low MOI to infect cells.
After the virus generation, customers can choose to authorize us to perform subsequent protein expression services. If you choose to terminate the service, we will deliver the virus stock to you in the first time. Please know that whenever you need help, Creative BioMart will always be there.