I'm trying to design an experiment to transfect Ewing Sarcoma cells with a plasmid carrying a targeted fluorophore. Since this is my first time trying to design an experiment like this, I was unsure if prior to transfection, and after my plasmid linearization with a restriction enzyme, would I need to remove the enzyme by purification or would I need to only heat-inactivate the enzyme? I'm only making a single cut as this plasmid was purchased from Addgene and should already have everything I need. Thank you in advance.
If anyone has any other suggestions or protocols they'd like to share, please feel free to let me know them!
Dear Erik,
Heat inactivation of the enzyme should be sufficient. However, if you try this and feel it is not working well, there are great kits from Qiagen (gel extraction kit or pcr cleanup kit) to purify DNA after molecular reactions and/or running on an agarose gel. Hope this is helpful.
Dear Erik,
Heat inactivation of the enzyme should be sufficient. However, if you try this and feel it is not working well, there are great kits from Qiagen (gel extraction kit or pcr cleanup kit) to purify DNA after molecular reactions and/or running on an agarose gel. Hope this is helpful.
Hi,
I think you should remove out the enzymes as it will increase the efficiency of your transfection. I usually remove it manually and it takes 2 hours before transfection step.
If you need a manual method, I can provide you in two simple steps or you can follow your lab kit. Good Luck.
Hello!
Is it obligatory for you to start with a linear plasmid? There is a risk to reduce the transfection efficiency (uptake is higher with coiled DNA). Nevertheless, it's better for stable transfection since integration into the genome would be easier...
Regarding your question, like Daniel I would suggest to clean up before starting. But depends on your cells and their transfectability also.
Best, Sandra
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