PCR fragments are prepared for LIC by treatment with T4 DNA polymerase.These are annealed into the appropriate vectors and used to transform XL1 Blue cells.Transformants are plated onto 48-well Q-trays and incubated overnight.Colonies are picked and used to inoculate 1 ml of media in each well of a 96-well block.These cultures are grown overnight and then plasmid DNA is purified by miniprepping in a 96-well plate on the Biomek.Positive clones are identified by PCR followed by Labchip analysis.
Guidance in our lab for PCR construction, processing for LIC, annealing, transformation and screening for positive clones, using the Biomek for minipreps and the Labchip for PCR analysis.
New Baculovirus Services:
Positive clones obtained as for E. coli expression are used to make recombinant baculovirus using the Bac to Bac system.Transpositions are carried out by transforming DH10 Bac cells in 96 well plates and then plating on indicator Q-trays.Transposition disrupts beta-galactosidase expression and is detected by blue/white colony selection. Recombinant bacmids are purified usingtraditional alkali lysis miniprep protocols.The bacmids are screened by PCR using M13 primers to confirm insertion of the gene of interest into the bacmid.Negative bacmids display a characteristic 273 base-pair fragment as the primary PCR product. The bacmid DNAs are used to transfect insect cells in suspension in 24-well blocks.Transfection in suspension has been shown to yield higher viral titers.The resulting virus is titered by QPCR.Virus titering by QPCR can be requested as an individual service.