Transfection efficiency is low.	Ensure that transfection occurs on actively proliferating cells at high density (~70–80% confluent). The ratio or mixing time of DNA and transfecting reagent may need to be varied, we suggest a DNA (μg) to P3000 (μl) of 1 to 0.5–5.0 and extending the incubation time to 20 minutes.
Many of cells are detached on the next day after transfection.	Varying hPSC lines demonstrate differing cytotoxicity to Lipofectamine; we suggest reducing the Lipofectamine volume by 2-fold.
No colonies appeared after antibiotic selection.	Either the puromycin concentration overcame the puromycin resistance of transfected cells or the cells were not efficiently transfected. Repeat the puromycin titration to ensure proper puromycin concentration and see troubleshooting for ‘Transfection efficiency is low’ above.
Colonies survived in negative control cells.	Repeat the puromycin titration to identify the lowest concentration which eliminates all control cells.
No colonies appeared after single cell plating.	Ensure use of StemFit Basic02, which allows for single cell expansion. However, differing cell lines demonstrate differing tolerance of single cell plating. Alternatively, we recommend plating the puromycin selected cells at low concentration in 6-well plates (10–50 cells/well) and picking individual colonies that were generated from a single parental cell. Place picked colonies in separate 1.5ml eppendorf tubes, wash with PBS, add 20ul of Accutase, and proceed with the outlined protocol.