The following cell line specific conditions are provided as a starting point for transfecting wild-type cells with TrueGuide Synthetic gRNA and TrueCut Cas9 Protein v2 using the Neon Transfection System 10 μL Kit. Further optimization of the electroporation or nucleofection conditions may be necessary for best results.

Cell type Source Media Number of cells/10-μL reaction (× 103) TrueCut Cas9 Protein v2/gRNA (ng/pmoles) Neon electroporation conditions*
Well format 24-well
HEK293 Human embryonic kidney DMEM 150 1250/7.5 1150 V/20 ms/2 pulses
U2OS Human osteosarcoma McCoy5A 150 1250/7.5 1400 V/15 ms/4 pulses
A549 Human epithelial lung carcinoma DMEM 120 1250/7.5 1200 V/20 ms/4 pulses
THP1 Human peripheral blood monocyte leukemia RPMI 200 2000/12 1700 V/20 ms/1 pulse (#5)
K562 Human leukemia bone marrow RPMI 200 1250/7.5 1700 V/20 ms/1 pulse (#5)
iPSC Human induced pluripotent stem cells Essential 8 80 1500/10 1200 V/20 ms/2 pulses (#14)
iPSC Human induced pluripotent stem cells StemFlex 80 1500/10 1200 V/30 ms/1 pulse (#7)
Human primary T-cell Healthy donor derived OpTmizer + 2% human serum 200 1250/7.5 1600 V/10 ms/3 pulses (#24)
Jurkat T-cell Human peripheral blood lymphocyte RPMI 200 1250/7.5 1700 V/20 ms/1 pulse (#5)
HepG2 Human hepatocellular carcinoma DMEM 120 1250/7.5 1300 V/30 ms/1 pulse (#8)
N2A Mouse brain neuroblastoma DMEM 100 1250/7.5 1400 V/30 ms/1 pulse (#9)
*Recommendations for the Neon electroporation settings are based on the culture conditions tested.