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foci formation assay - protocol (Aug/04/2009 )


Has anyone done foci formation assay, if so, I would appreiate protol sharing. I would like to do foci formation assayon PLC cell line. I don't believe the protocols written in journals as half the time the infomation is incomplete and they miss out the most commonly done mistakes in any given experiement.

am'I correct in my protocol:

Seed cells in 6 well plates (each sample in duplicate): PLC vector-control cells and PLCs-transfectec with gene of interest.
Transfect the PLC cells with your gene of interest and select cells next day with antibiotc selection. But for the vector control cells just add normal medium without antibotic. Change medium every 3 days and stain with crystal violet after 2 weeks?
Is it best to do in 6 well plates?



You should also have an off target selection with no transfection, just to prove that your cells will die with the concentration of antibiotic and within your timepoint