Edited by littlecell, 04 March 2005 - 08:26 AM.
Posted 04 March 2005 - 08:26 AM
Posted 04 March 2005 - 08:51 AM
It's also worth noting that transfection effciency and conditions vary between cell lines for example a neuroblastoma cell line I use requires about 10 times as much DNA and more lipfectamine 2000 than HEK293s. It might be worth titrating the DNA and lipofectamine amounts to see what happens
Hope this helps
Posted 06 March 2005 - 05:39 PM
Posted 06 March 2005 - 08:03 PM
I think the answer to the initial question is that the cells should be passaged so as to get actively growing/dividing cells as these are more likely to take up the DNA and express it. If you have confluent or nearly confluent cultures then the cells will be starting to or more likely to senesce which means that none of your DNA will be taken up or expressed!
I too am fairly new in this area, but this is the explanation I have been given by others for splitting the cells before transfection.
Posted 07 March 2005 - 12:55 AM
post doc2130 and BOB1 are right.
For best resutls cells should be active (growing/dividing) and as you need to test transfection conditions regarding confluency, you should seed plates with different conditions and test them. But as neuromatt said, these will partially depend on the transfection agent you use.
Edited by fred_33, 07 March 2005 - 12:56 AM.
Posted 08 March 2005 - 07:45 AM