Organization of the BV expression service
Starting materials to be provided by user
Computer Records
The baculovirus computer records can be found in the Baculovirus folder, in the Barford & Pearl folder, on our shared network drive.
| Contents of 'Baculovirus' folder | Description |
| BV_progress.xls | Table indicating status of viruses being prepared. |
| BV_expression.xls | Table summarising expression and crystallisation results for BV proteins. |
| Names of viruses | Folder containing protein family sub folders, and excel files listing the names of constructs made for each individual protein. |
| Other virus data | Folder containing BV_Fridge1.xls, BV_Fridge2.xls, BV_Fridge3.xls and Day2_BVs.xls. |
Please help us keep the BV_expression.xls table up to date by adding new data as it becomes available.
| Contents of 'Other virus data' folder | Description |
| BV_Fridge1.xls | Technical data and locations of viruses (pre 2001) |
| BV_Fridge2.xls | Technical data and locations of viruses (2001-2003) |
| BV_Fridge3.xls | Technical data and locations of viruses (2003-) |
| Day2_BVs.xls | Names of viruses which produce optimal amounts of protein after a 2 day infection. |
Description of service
Vivienne and Sara will prepare up to 12 viruses at a time using standard protocols.
If you are working with a collaborator, please ask preferably for a titered virus. It will generally only take us about 2 weeks to produce protein from a titered virus. Please send a completed BV submission form (available as a Word or PDF document) to Vivienne.Thompson@icr.ac.uk or Sara.Kisakye-Nambozo@icr.ac.uk. If you give us an untitered virus, please allow another week for titering. If you give us a bacmid, please allow a full month for 2 rounds of amplification and corresponding plaque assays.
If you provide us with a recombinant pFastbac plasmid, please allow 2 months from the "Date work started" before you book your first expression experiment. This will allow us to do the transpositon, bacmid preparation, confirmatory PCR, transfection and viral amplifications. Progress of your virus can be checked from the BV_progress.xls table, available in the Baculovirus folder in the Barford & Pearl folder, Shared on Holyhead. Please note, however, there will be no bacmid preparation service when either Vivienne or I are on holiday.
It is important that you book your expression requirements at least 10 days in advance, otherwise we may be growing very expensive Sf9s that are not needed.
When we harvest the cells for you, we will also take a small sample (3.3 x 105 cells ) to be lysed in 50µl 1xSDS GLB. This will be stored in the -20°C freezer, top shelf in 5C8. The location of your sample can be found from a laminated chart located on the freezer door. Please wipe off the entry for your pellet when you remove it, and throw away anything not required. When we have time available, we will carry out gel and blot analysis on expression samples. However, we recommend that you carry out your own analysis if results are required urgently. To do this, heat your sample to 95°C for 3 mins before loading 10 µl onto an SDS-gel for analysis. Negative control Sf9 gel samples are available. The remainder of your infected cells are spun down and stored in Falcon tubes at -80°C. The pellets are located in a metal tray at the bottom of freezer A.