6 replies to this topic

[jzhen]

Hi, all:
  I am just starting to get familiar with transfection. To the media for trasnfecting the cell lines, could I add penicillian/ streptomycin in?
  If not, after transfection, could I add them in? What is a good time to add P/S?
  Thanks ahead
  Juan

[geness]

hi,
   I  am in the same situation with you!
   When I transfect the cells the first time,I add P/S into the media but  the transfection efficiency was very low.So the second time I do not use P/S in the media for my transfection,to my disappointment,the transfected cells were contamined mostly .

   If someone know something about this,please tell us.
   Thanks!

Edited by geness, 14 April 2004 - 06:58 AM.

[kant0008]

Hi guys!
I add pen/strep to all my cultures, including transfections. You're better off in most cases with a lower transfection efficiency than no cells at all due to contamination. Some transfection reagents/methods work better with some cells and not others, so I suggest use pen/strep but maybe look at other transfection methods.

[geness]

Thank you very much!
   I agree with you!By the way,which method did you select?(chemical or physical) and I use lipofection  for my transfection.I am giong to try to use CaCl2,but I wonder whether the transfection efficiency is satisfing.

[jzhen]

geness, on Apr 15 2004, 03:04 AM, said:

Thank you very much!
   I agree with you!By the way,which method did you select?(chemical or physical) and I use lipofection  for my transfection.I am giong to try to use CaCl2,but I wonder whether the transfection efficiency is satisfing.

I agree too. I am using lipofectamine, which I don't know whether is the same method with lipofection.  In my case, I use G418 to select the transfected cells, which has anti-G418 gene, transfected together with the gene of interest.

  I was told that P/s does have some effect on the transfection. Someone suggest me that except the few hours of transfection, I should use P/S all the time.

[kant0008]

Hi!
I tried Calc Phos, but it didn't seem to be particularly good. A similar methodology, but using Dextran sulfate normally works better, but there are problems with toxicity, so you got to watch it. Lipofectamine seems quite good, but you might still try smth different, like Fugene6 or DOTAP, there are tons of different reagents on the market now.

[fred_33]

Hi. For now, i've done transfection in 293 HeLa and endothelial cells. I use lipofectamine and do not add Peni/Strepto to the media for the transfection. I let the thigns happen for 3 or 5 hours (depend of the cells) then i add a complete media (ie with peni strepto) with twice the normal concentration of serum (only required if you want contihnual growth).
I never get contamination.