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Maintaining stable cell line - (Feb/14/2008 )

Hi, I have made transient transfections of PC12 cells with vector-GFP construct and shown that GFP is expressed. Now, how would one make a stable cell line? From my understanding, you have to grow the cells in the presence of G418. However, it's not clear whether or not I should passage these cells in between or not. Any suggestions or tips would be appreciated. Thanks.

-qkchen-

we do 3-4 passages of cells in the G418 (or any other antibiotic) and finally confirm the expression of desired protein by western blot.

-scolix-

QUOTE (scolix @ Feb 14 2008, 02:12 PM)
we do 3-4 passages of cells in the G418 (or any other antibiotic) and finally confirm the expression of desired protein by western blot.


how long is your entire selection procedure?

-qkchen-

QUOTE (qkchen @ Feb 15 2008, 01:24 PM)
QUOTE (scolix @ Feb 14 2008, 02:12 PM)
we do 3-4 passages of cells in the G418 (or any other antibiotic) and finally confirm the expression of desired protein by western blot.


how long is your entire selection procedure?


depends on cells (cell line, kind of transformation), and G418 concentration; my experience is: better start with not too high (<800 µg/ml) G418 and select over 2 weeks than with high concentration (>800 µg/ml)

-The Bearer-

QUOTE (The Bearer @ Feb 15 2008, 01:32 PM)
QUOTE (qkchen @ Feb 15 2008, 01:24 PM)
QUOTE (scolix @ Feb 14 2008, 02:12 PM)
we do 3-4 passages of cells in the G418 (or any other antibiotic) and finally confirm the expression of desired protein by western blot.


how long is your entire selection procedure?


depends on cells (cell line, kind of transformation), and G418 concentration; my experience is: better start with not too high (<800 µg/ml) G418 and select over 2 weeks than with high concentration (>800 µg/ml)


thanks, that was helpful. what about selecting for a single clone of transfected cells? or should i use an entire population of transfected cells for post-translational modification of protein study? thank you.

-qkchen-

QUOTE (qkchen @ Feb 15 2008, 02:24 PM)
QUOTE (scolix @ Feb 14 2008, 02:12 PM)
we do 3-4 passages of cells in the G418 (or any other antibiotic) and finally confirm the expression of desired protein by western blot.


how long is your entire selection procedure?



Its around 2-3 weeks.

-scolix-