If anyone could help, it would be greatly appreciated. I am currently in the middle of introducing a targeted knockout in human embryonic stem cells - I am using the plasmid px459v2 which has has my gRNA cloned inside it.
Furthermore, I am using lipofectamine stem. In terms of methodology, I waited for the cells to be confluent and at which point, I introduced the transfection reagent along with the plasmid as suggested by the thermo protocol available online. I then proceeded with a 3 day puromycin selection at 1ug/ml. At the end of the 3 days, I noticed that there were some single cells remaining and I have been culturing those single cells with CloneR + mTEsR. But its been 4 days and I see no real change in terms of growth. Is there something im doing wrong or is electroporation the answer?
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Hello Smarth,
I don't have experience with hESCs, but I do have some experience with optimization of transfections and what I can suggest is the following:
- Make sure to test different concentrations of lipofectamine ( n and 2n p.e.), or even try asking a colleague for another transfection reagent to compare with. For me, for instances, some cells do well with any transfection reagent, while other had better efficiency using fugene HD instead of lipofectamine.
- Try to transfect at 40-60% confluency
- Make sure to have kill curve of puromycin for your cells, as sometimes, it can be too high concentration.
You can be observing low transfection efficiency, which then, after selection, you end up with too few cells, for them to signal properly and stimulate proper growth rate.
Hope it helps
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