When do I add selection?
Posted 29 November 2010 - 08:08 AM
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!
Posted 29 November 2010 - 11:48 PM
Posted 30 November 2010 - 02:21 PM