2 replies to this topic

[andyg2886]

Hi all

As a PhD student doing a lot of cloning work (through electroporation) I have a question that keeps popping up but which everyone seems to have a different answer for. Therefore, I thought I'd pose it to the forum to see if a consensus answer can be reached.

I work with two different types of cell lines - human embryonic stem cells, and cancer cells.

I am transfecting GOI's in the pCAG vector (harboring the puromycin resistance gene) into both the ES cells and the cancer cells. They are very different cell lines - ES cells grow in colonies whereas the cancer cells grow without many problems from single cells.

The question: How many hours after the electroporation do I add puromycin?

I've had a range of answers from 'when to well is completely confluent' to 'when the well is 80% confluent' to 'add as soon as the cells have settled on the bottom of the plate'

It seems sensible for  the ES cells to add puro as late as possible - they rely on colony-dependent growth so is it sensible to let them grow to the colony stage first? As for the cancer cells - ASAP or what?

Let me know your thoughts!

Cheers

Andy

[Rsm]

Puromycin is toxic to the cells, so when you add imediately after plating, you will kill all cells, also the correctly targeted ones which do not yet express the puromycin resistance gene. I always wait for 48h, you will anyway have lots of dead cells after electoporation. And 99.9% of your cells will not have the plasmid, so you will kill almost all with puro after 24h treatment. So no problem with overgrowth...


rsm

[bob1]

Most selective antibiotics require that the plate be no more than 70% confluent for them to work effectively.  Do not add the selective agent immediately after transfection.  Most protocols add selective agent after 24 hours.