Help!... Stable transfection.. - A293T with puromycin gene.. (Sep/07/2005 )
I'm a little bit in the dark here, so I was hoping people could help me out here. I have done an electroporation of A293T cells with my gene of interest en the puro gene. And it seems to have worked.. In my no DNA control all my cells are dying and in the flask with the gene I have some survivors.. Jeuheu...
But.. my cells (adherent) are in a flask.. And I'm not sure if I can change the media.. and trypsinize them.. It probably couldn't really hurt them, but I wasn't sure.. because I totally lack any experience in this.. So please tell me. How long do you wait to change the media or to trypsinize the cells? It's been 6 days now (after adding the puromycin, so 7 days after electroporation) and the ones with the DNA seem to divde also.. I really need to get them to a plate... I don't think it could hurt.. but.. I just wanted to be sure...
Thanks for the reply!
You can definitely change the media and trypsinize in flasks just like dishes. In fact, in my lab we use nearly exclusively flasks for all our tissue culture. I would recommend you change the media soon. Normally puro selection is complete after a few days. Also 293s generally like to be fed and passed pretty regularly.
I suggest you do the following for passaging:
- Remove cap and use a sterile aspirating pipet to remove all media.
- Wash once with PBS and remove PBS.
- Add a small volume of tryspin and rock back and forth.
- Close cap again and put back in incubator for a few minutes until up from flask.
- Remove cap and add several mLs of serum containing media.
- Pipet up and down and pass as desired to new flasks etc.
If you want to feed the cells merely gently pipet in the desired volume after removing the cap and aspirating the old media. Replace the cap once the new media has been added.
Also you should know that 293T cells transfect very well with standard lipofectamine methods. We do not find it necessary to go the electroporation route.
293 cells grow quickly and you should change the media every two days. Don't hesitate to trypsinise your cells. They can survive it.
I usually dilute in PBS at 1/5 the trypsin and let it 5' at 37°. After, rock back and forth. Add great amount of medium. Centrifuge 5' 200g.
Remove sumernatant and resuspend cells, and transfert in plates / flasks (generally 1/3 or 1/5 dilution.)
Thank you guys.. You have helped me a lot. I will pass them in the next hour!