Hello,
I Wonder why we have to heat inactivate restriction enzyme before transfecting cells with a linearized plasmid with Lipofectamine (3000) to establish stable cell lines. What if my enzyme is not activated since it will be diluted in the optimem, medium and since my plasmid has only one restriction site?
Do you think that the restriction enzyme can enter the cytosol and cut the ADN?
Does it have a real impact ? Or is it a kind of "thing people are used to do"?
What do you think?
Thanks
Mostly we do the heat inactivation of the restriction enzyme when we are doing cloning. Heat inactivation of the restriction enzyme is most critical step before proceeding to ligation, followed by transformation.
But in this case the only reason I can think is that presence of the restriction enzyme might somehow affect the transfection efficiency of the cell line.
Thanks for your answer Chandhuru,
I think that the conséquences are much evident when you do ligation for transformation, but i really think that it has an impact when you transfect and select clones for stable transfection.
The problem is that it seems difficult to see how much downstream functions may be disturbed by the restrction enzyme in your transfected cells.
Have a good day.
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