selecting single cell colonies using single cell per well dilution one day after - (Nov/04/2006 )
I am transfecting my V79 chinese hamster cells using lipofectamine 2000. In lipofectamine transfection protocol you do the transfection when the cells are nearly confluent and activly growing, then one day after transfectin you add the meida plus antibiotics to the cells. As i have to pick up many single cell colonies from the transfected colonies, and test them with restriction digest and southern blotting for right integration of the plasmid, and choose the right one, can i do the single cell/well dilution for example 1 cell per 100 microliter in each well of a 96 well plate.
and do the selection after this? can a single colony survive at least 10 days of selection period in hygromycin selection in a single well?
-abyasemani-
QUOTE (abyasemani @ Nov 4 2006, 11:18 AM)
I am transfecting my V79 chinese hamster cells using lipofectamine 2000. In lipofectamine transfection protocol you do the transfection when the cells are nearly confluent and activly growing, then one day after transfectin you add the meida plus antibiotics to the cells. As i have to pick up many single cell colonies from the transfected colonies, and test them with restriction digest and southern blotting for right integration of the plasmid, and choose the right one, can i do the single cell/well dilution for example 1 cell per 100 microliter in each well of a 96 well plate.
and do the selection after this? can a single colony survive at least 10 days of selection period in hygromycin selection in a single well?
and do the selection after this? can a single colony survive at least 10 days of selection period in hygromycin selection in a single well?
dilution to statistical seeding of 1 cell per well is fine; for recovering you may discontinue application of antibiotics, and add 2 or 3 days later
-The Bearer-