question about plasmid transfection - (water solution or buffer solution?) (Mar/02/2007 )
I need to transfect some cells with plasmid. The plasmid stock solution in our lab is usually 50ng/ul in water. According to Invitrogen's Lipofectamine 2000 user manual, 4ug per well should be transfected in 6-well plate. That would need 80ul the plasmid stock solution. I am wondering if the water solvent will affect the cells. Should I exchange the plasmid to some buffer solution, e.g. PBS or Tris (pH 8.0)?
Any advice would be really appreciated!
It seems you dont have a maxi prep of the DNA. Better make one its easier for transfections.
I use only 2ug of DNA for 1 well in a 6 well plate.
The DNA in water shouldnt affect your cells for transfections. I would suggest precipitating the DNA and concentrating it either water or TE for transfections.
Precipitate your DNA,I do it all the time. (I have to linearise my DNA so it comes in relatively large volumes, so I have to reduce the volume as well as ions etc, so I precipitate). Works for perfect LF2k, electroporation and calcium-phosphate. Just make sure to remove all Ethanol.
I use 1ug of Qiagen column purified DNA per well of 6 well plate which works fine. Its better to do a midi or a maxi and keep an aliquot ( ex:1ug/ul) for regular use. DNA in water has worked fine for me.