making a stable cell line - (Jun/24/2008 )
when making a cell line, how often do you passage your transfected cells? or do you just throw G418 on the transfected cells and wait for them to grow to a sufficient density before passaging? thanks very much.
-qkchen-
QUOTE (qkchen @ Jun 24 2008, 01:17 PM)
when making a cell line, how often do you passage your transfected cells? or do you just throw G418 on the transfected cells and wait for them to grow to a sufficient density before passaging? thanks very much.
I,
1. replace the G418 every other day.
2. do not passage the cells.
3. let them grow to become about 0.4cm size islands, and then use cloning ring to trypsinize and transfer individual clones to 12 well/6 well plate.
-cellcounter-
QUOTE (cellcounter @ Jun 24 2008, 03:19 PM)
QUOTE (qkchen @ Jun 24 2008, 01:17 PM)
when making a cell line, how often do you passage your transfected cells? or do you just throw G418 on the transfected cells and wait for them to grow to a sufficient density before passaging? thanks very much.
I,
1. replace the G418 every other day.
2. do not passage the cells.
3. let them grow to become about 0.4cm size islands, and then use cloning ring to trypsinize and transfer individual clones to 12 well/6 well plate.
how should i modify your protocol if my supervisor wants a pool of transfected cells and not an individual clone.
-qkchen-
QUOTE (qkchen @ Jun 25 2008, 10:08 PM)
QUOTE (cellcounter @ Jun 24 2008, 03:19 PM)
QUOTE (qkchen @ Jun 24 2008, 01:17 PM)
when making a cell line, how often do you passage your transfected cells? or do you just throw G418 on the transfected cells and wait for them to grow to a sufficient density before passaging? thanks very much.
I,
1. replace the G418 every other day.
2. do not passage the cells.
3. let them grow to become about 0.4cm size islands, and then use cloning ring to trypsinize and transfer individual clones to 12 well/6 well plate.
how should i modify your protocol if my supervisor wants a pool of transfected cells and not an individual clone.
yes, ppl have done that. Just trypsinize all you have and replate to large size plates then pass as usual.
-genehunter-1-