Question

A researcher attempted to transfect HEK293 cells with the Influenza A H1N1 PR8 Hemagglutinin expression plasmid using PEI MAX as the transfection reagent. However, SDS-PAGE and CBB staining did not show any bands representing hemagglutinin, unlike the negative controls. Which aspect of the protocol might be a potential issue?

A) The concentration of the plasmid used for transfection was too high.
B) The concentration of PEI Max used for transfection was too low.
C) The incubation time after transfection was insufficient.
D) The cell confluence before transfection was too high.

Answer 1

A failed transfection attempt using PEI MAX on HEK293 cells could be due to issues with plasmid or PEI MAX concentration, insufficient incubation time, or high cell confluence. Troubleshooting would require adjustments to these parameters to identify the problem.

Step-by-step explanation:

The question pertains to a failed attempt to transfect HEK293 cells with the Influenza A H1N1 PR8 Hemagglutinin expression plasmid using PEI MAX as the transfection reagent. The absence of detectable bands representing hemagglutinin on SDS-PAGE and CBB staining suggests potential issues with the transfection protocol.

1. The concentration of the plasmid used for transfection being too high could lead to toxicity or the formation of aggregates that may be difficult for cells to uptake effectively.
2. Insufficient concentration of PEI Max could have led to inadequate complexation with the DNA, reducing the efficiency of transfection.
3. The incubation time after transfection might have been too short for the expression and accumulation of detectable levels of the protein.
4. Cell confluence before transfection is a critical variable as well. Optimal transfection requires a balance; too high confluence can limit transfection efficiency due to diminished cell growth and division.

Each of these factors is important to consider when troubleshooting transfection experiments. Modifications to these parameters and additional control experiments could help identify the specific cause of the failure.