stable cell line - how it work? (Nov/15/2006 )
stable cell line can be obtain after transfection of the cell with a plasmid containing a G418.
for exemple Ihave a GCP g418 plasmid
How it works? after tranfection you plate the cell in medium containing G418 then you check fluroscent cell and you keep doing the propagation? how long it take to get a stable cell line.
how do we do with a plasmid with no GFP?
thanks
jm
Run a control with untransfected cells in parallel do see when those cells are killed by the G418, then most likely transfected cells that are surviving will be stable transfections.
First of all you have to transfect the cells and then wait for som time. After that, I usually make it after 2 days after transfection I put the cell to the other bigger plate and put the medium with G418 (Neomycin)... Change up to time medium, don't forget about G418. After week or so the cells would be dead and u will see some clones... After 2-3 weeks you could pick up the colonies and put them to 96 or 24 well plates grow them up and then freez. So it is in a short way...
If you have fluorescence - your life is quite easy - pick up the colonies only with fluorescense...
If not - pick them all, and then u have to make Western Blot, or Immunostaining to be sure that your gene is expressed (overexpressed)... Usually u make it with plasmids which have CMV, EF1 or other promotor for overexpression...
The problem with NeoR is that G418 (Neomycin) gives a lot of false-positive colonies... That is because of integration in genome only of NeoR cassette, and full of your plasmid... Some people make linearization of vector before transfection - they claim that it helps to have full integration in genome... I don't blieve it, so make it only with plasmid... But have to say that sometimes I have only 1 from 30 colonies which is positive, so to claim that all colonies which are survives are posotive is not correct! But it always depends on your construkt, your cell lines and your luck! ;-)
If you have more questions - read some protocols, how ppl make it... Before starting would be also nice to test concentration of G418 in your case for your cells and your charge of G418... There are some information - I think also in instruction for G418... I usually use 400-500 ug/ml for HeLa cells... For me it works nice... You could also check literature in pubmed about ur cell lines and wich concentration ppl use in their previos works...
Don't forget!!! If your plasmid doesn't have NeoR casette it possible that you have to use other antibiotik (e.g. Puromycin, Hygromycin or Zeocin)
[quote name='Fedo' date='Nov 16 2006, 07:07 AM' post='77253']
[quote name='tap14' post='77095' date='Nov 15 2006, 08:19 PM']
Run a control with untransfected cells in parallel do see when those cells are killed by the G418, then most likely transfected cells that are surviving will be stable transfections.
[/quote]
First of all you have to transfect the cells and then wait for som time. After that, I usually make it after 2 days after transfection I put the cell to the other bigger plate and put the medium with G418 (Neomycin)... Change up to time medium, don't forget about G418. After week or so the cells would be dead and u will see some clones... After 2-3 weeks you could pick up the colonies and put them to 96 or 24 well plates grow them up and then freez. So it is in a short way...
Hi thanks
when you say "pick up colonies and put them in 96 or 24" how do you do that ? I mean litteraly how do you take a colonie and transfert in other well in a steril manner?
thanks
anyone?