TABLE 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
1, 30, 39, 43, Boxes 1 and 2	Change in media color and/or presence of foul smell	Contamination of cell culture due to improper sterile technique	Ensure proper sterile technique is used. Clean and disinfect hood work surfaces before and after every manipulation. Before working in the hood disinfect gloves with 70% alcohol. Spray all items to be placed in the hood (media bottles, pipetting devices, outside of the plastic wrap the sterile containers, etc) with 70% ethanol and wipe them with paper towels.
		Contaminated virus	Ensure virus has been filter sterilized.
		Contaminated reagent	Filter sterilize histone deacetylase inhibitors (i.e. sodium butyrate) in the hood.
38	Low virus titer	Sf9 cells infected at the wrong MOI	Generate P2 virus using lower MOI (0.05–0.0001).
			Remake the bacmid ensuring that the bacmid culture is inoculated from a white colony and that the bacmid DNA is not sheared during purification.
45, 50	Low cell harvest density	Poor-quality cells	It is important not to let cells overgrow (i.e. to subculture them on a regular basis) and to avoid using cells that have undergone more than 30 continuous passages since being raised from liquid nitrogen. Check health and viability of cells prior to each experiment.
	Low expression	Infection at the wrong MOI (i.e. too little or too much virus), non-optimal expression conditions, toxicity of membrane protein when overexpressed	Always determine the titer of the BacMam virus to caculate a MOI. Screen for optimal temperature, harvest time and concentration of histone deacetylase inhibitors.
		Virus is not stable when stored for extended periods	Remake the P2 virus starting from either the P1 virus or a glycerol stock of DH10Bac E. coli containing bacmid DNA. Use TIPS method66 to preserve Sf9 cells infected with P1 virus.