pcr product transfection help - (Jul/25/2006 )
i would like to clone a pcr product directly into animal cell lines. am afraid if the cmoponents in serum wud degrade my product even before transfection.
has anyone done this before?
what are the precautions to be taken?
I do combined transfection of cut plasmid and PCR product (for recombination). Don't take any other precautions than I would for plasmid transfection. Just make sure your PCR product is cleaned after the PCR.
wat method do u follow?
we have lipofectamine 2000 as well as capo4 mediated methods.
wat should be th edna concentration?
I'm doing Calcium phosphate on 293T's, buf have in the past done the same with LF2k on Hela cells. I'm not too sure about the DNA concentration actually, but I'm just taking the total "standard concentration" (vector + PCR product together equal standard concentration for the applied method and cell line). Maybe perform a control reaction with EGFP PCR product (well, a promoter and EGFP).