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This maybe a stupid question, but i just wonder why you want to check the resistance against g418 in hek293t cells This time the cells all survived the night, were confluent and therefore split into a small flask. The reason why i ask is that i am assuming that you want to make stable cell line in 293t (if not, then just ignore me, please).

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The seem to not plate very evenly The first time all cells (nih3t3) died over night after virus was added I would say the problem has gotten worse and worse the more we try different things.

3) i did not change the media the day after transfecting 293t cells.

There seems to be no pattern in the way they behave, sometimes they attach and grow and sometimes they don't. Good news, my antibody works pretty well (tested 293t untransfected, transfected 293 (two different clones), and psirpg transfected cells (transfection control). To transfect my 293t cells, i have been using fugene 6 That has worked fine for me

I then harvested the supernatant, which i used to transduce 3t3 cells, in the presence of polybrene to determine the transduction efficiency In the past, this also has worked fine Recently, i have had some problems, and am trying to think of things that i have altered Regarding the hepes addition, i meant.

I use them to produce aav and lentivirus

Anyway, i bought these from atcc and they arrived today The documentation says they are at passage number 17. I tested several transfection reagents at the same time (fugene hd, mirus, jetpei) in 293t cells and found out that gfp expression was highest in 293t cells transfected with jetpei.