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problem in plasmid transfection


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#1 sososun

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Posted 13 May 2010 - 04:36 PM

Hi All,
I am facing a problem with pretty low transfection effeciency in HeLa cells (around 2-3% of green cells). The vector is eGFP-N3, and my insert is about 3.5kb.
I used lipofectamine 2000 at a ratio of 2.5:1 to plasmid DNA (2ug/well in a 24-well plate). The procedure was done based on the manufacturer. Medium was replaced after 4-5 hours. However, after 24h transfection, many cells were dying, and most of the cells seem unhealthy. Very few cells were green. After another 24h, more than 60% cells died, and most green signals were from those dead cell debris. by the way, the initial cell density was around 90%.
I tried to use Fugene HD, but the efficiency was still as low as with lipofectamine 2000, although not so many cells died.
Could anyone help me troubleshoot? Many thanks!
best
frank

#2 bob1

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Posted 13 May 2010 - 04:44 PM

GFP constructs can be very energy intensive for cells as they express very highly and use a lot of cell resources to produce an essentially useless protein. Try using less DNA (and correspondingly less lipofectamine) - the Lipofectamine 2000 manual recommends 0.8 ug/well for 24 well plates.

#3 sososun

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Posted 13 May 2010 - 05:15 PM

GFP constructs can be very energy intensive for cells as they express very highly and use a lot of cell resources to produce an essentially useless protein. Try using less DNA (and correspondingly less lipofectamine) - the Lipofectamine 2000 manual recommends 0.8 ug/well for 24 well plates.


Many thanks!
what I used was 6-well plate. (sorry for the mistake). I am going to use 24-well plate since the reagent is running low.




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