flashBAC Manual - Oxford Expression Technologies

5 Incubate the dishes at room temperature for 1 hour ona level surface for virus adsorption. Do not leave thedishes for longer than 1 hour (40 minutes is the minimum).• It is important to ensure that the cell monolayer does notdry out at this stage. If working within a Class IImicrobiological cabinet, remove the dishes to the benchat this stage to prevent the cells drying out.6 About 15 minutes prior to the end of the virus adsorptionperiod, prepare the LGT agarose overlay. Completely melt1 x 10 ml aliquot of ready-prepared and solidified 2% (w/v)LGT agarose (in a microwave oven or boiling water bath,taking appropriate safety precautions) and, after cooling toabout 50°C (hand-hot), add an equal volume (10 ml) ofappropriate insect cell culture medium. Mix thoroughly butgently, avoiding air bubbles.• You need 2 ml of this agarose/medium overlay for eachdish.• Use immediately or keep warm at 45°C to preventsolidification. If using a water bath, ensure it is cleanand wipe the bottle of overlay with alcohol before use toprevent fungal/bacterial contamination of the plaqueassay.• If using antibiotics, add to the culture medium beforepreparing the overlay.• Should the agarose set before using, do not re-melt it;prepare a fresh batch.7 After the virus adsorption period and after preparing theagarose overlay carefully remove the virus inoculumOXFORD EXPRESSION TECHNOLOGIES 34