TABLE 2.

Troubleshooting

Step	Problem	Possible reason	Possible solution
10	The concentration of empty pBeloBAC is low	Bacterial culture is inappropriate	Grow the bacteria at 37°C for at least 16 h with 250-rpm shaking in an orbital shaker
		Cell lysis is not complete	Resuspend the bacterial pellet thoroughly in solution I and let the bottle stand for 5 min after adding solution II and mixing gently
		The wash buffer is not prepared correctly	Check that ethanol has been added before using
18	Smeared bands	Plasmids have degraded	Store the pBeloBAC plasmids at −80°C
		DNase contamination of the buffer	Use a newly opened buffer
25	No colonies found after electroporation	Use of incorrect LB agar plates	Check that LB agar plates containing chloramphenicol are being used
		Contamination of residual ethanol left in the ligated DNA	Ensure that the ethanol is completely removed before resuspending the ligated DNA in nuclease-free water
		Bacteria have died after electroporation	Add SOC medium immediately after pulsing
38	Significant cell death after transfection	Low quality of the plasmid maxiprep preparation	Follow the maxiprep procedures carefully, particularly the washing step with HBC buffer
		Too few cells were used for DNA transfection	Seed a minimum of 5 × 105 cells/well for transfection. Increase the no. of cells/well if needed
		Too much Lipofectamine 2000 was used	Use no more than 10 μL of Lipofectamine 2000 for each well Change the transfection medium for postinfection medium at 6 h posttransfection
65	Undesired mutations occur in pUC57-F1/Venus-2A	Mutations occurred during PCR	Always use high-fidelity DNA polymerase for PCR amplification of the DNA
		Mutations occurred during propagation of Escherichia coli	Use NEB stable competent cells for transformation
91	No fluorescence observed in transfected cells	The pBeloBAC-FL/Venus-2A plasmids are inappropriately prepared	Check that the concentration of pBeloBAC-FL/Venus-2A is adequate
		Transfection has failed	Include a plasmid expressing a fluorescent gene under a constitutive polymerase II promoter as an internal transfection control
96	No fluorescent plaques observed	The cells are killed by the semisolid medium	Overlay the semisolid medium when it is at 42°C or less
		Supernatant is overdiluted	Infect the monolayer cells with a dilution of 1:10