I have tried to use Lipofectamine 3000 reagent to do transfection, but I encountered some problem that when add plasmid into Lipofectamine3000 Reagent I got cotton-like sediment leading to low transfection efficiency. I thought this segiment is DNA but why? I performed the mix step at room temperature and the OptiMEM was prewarmed to 37℃. How can I solve this problem?
Dear XaoYu, mixing DNA with cationic polymers may result in high MW aggregates which precipitate or form visible crosslinks. The order and speed of mixing and to a certain extent, the concentration of the solutions, will affect the result.
Assuming the manual proposes to add DNA into the reagent, you might try to vortex the diluted reagent while adding the DNA slowly.
No need to pre-warm the medium, when dealing with such small volumes. It will be back to room temp, when you start to pipet the complexes onto your cells.
Thank you so much for your answer and the advice you provide. I have tried as what you mentioned and the problem was solved.
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