tell me what im doing wrong? transfection - (Oct/23/2007 )

dear all,

im currently working on transfecting my HEk cells, using fugene method. My tec plasmid is approximately 4,9kb.
Ratio of fugene : dna used is 3 : 1. (Dna 2µg)
Im seeding my cells at a conc of 400, 000 cells/ ml per well in a 24 well plate. This is done a day before the transfection itself.

i dont know why my transfection rate is so low only 10 percent!!!!
please help

thanx

dear all,

im currently working on transfecting my HEk cells, using fugene method. My tec plasmid is approximately 4,9kb.
Ratio of fugene : dna used is 3 : 1. (Dna 2µg)
Im seeding my cells at a conc of 400, 000 cells/ ml per well in a 24 well plate. This is done a day before the transfection itself.

i dont know why my transfection rate is so low only 10 percent!!!!
please help

thanx

i harvest my cells..2 days after transfection..
.i get aprroximately 80 percent survival. I did western blotting to check on my protein expression..hoping to see an upregulation as compared to untransfected cell...
but nothing convincing..

I suppose my transfection is not working...and i check this by transfecting my plasmid together with GFP ...just to get an idea about the efficency....

Maybe you put too much cells when you did transfection? It should be 60-80% confleunt, or 1/3 empty space between cells.

That might be one of the reason...
I seed 400,000 cells/well...in a 24 well plate.one day prior...

do u think this is too much? cos its only 80 percent confluent on the day of transfection...its driving me nuts

How dense are the cells just before transfection? If they are too dense, the efficiency will go down.

meaning?


before i put them in the wells...
well...in the culturing bottle..its fully grown before i put them in the well...
and the day of transfection ..its about 70 - 80%

Try to plate cells so that they are a bit less dense, like 60-70% on the day of transfection.

also verify the DNA is clean or transfection grade. Else it will not work.

I have never tried fugene before but if you are sure about the fugene/DNA ration so its always the cell condition.
try to use OPTI-MEM media for transfection and for your cells NEVER to let it grow to 100% confluent in ur flask or plate if you are planning transfection on monday for example try to seed your cells in low density so at monday morning your plates are 70% confluent or so * i prepare extra dishes * to start seeding your transfected cells .