Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/05/29
|K562 Neon Transfection Results, Day 2||Main project page|
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Today I'll be checking the transfection efficiency of the neon transfection system on K562 cells using fluorescence microscopy and flow cytometry, then moving forward from there depending on the results.
Note: Even numbered wells were replicates of the preceding wells. None of them showed significant transfection efficiencies. This is due to pipetting error during the neon transfection protocol - there wasn't enough of the DNA/cell mixture to completely fill the pipette tip for the duplicates. Next time fix this by incorporating enough of a buffer for there to be some cells/DNA left over after all replicates.
Counts are low due to low cell density and volume (only 50 µL of each sample was used to measure transfection efficiency). Despite that, initial results look promising. Methods 2 and 3 both have high transfection efficiencies for pMaxGFP (95% and 90%, respectively) and KAH126-MV2 (12% and 21%, respectively). Will have to repeat using higher concentration KAH126-MV2 plasmid; 809 ng/µL is a little on the low side.
There's also a lot more debris / dead cells in the cultures treated with methods 2 and 3. Need to followup with a cell viability assay in the future as well.