Neon Transfection Conditions

Link to Life Technology's Neon Transfection System

Optimization Conditions


Condition Number
Pulse Voltage
Pulse Width
Pulse Number
 1 0 0 1
 2 1400 20 1
 3 1500 20 1
 4 1600 20 1
 5 1700 20 1
 6 1100 30 1
 7 1200 30 1
 8 1300 30 1
 9 1400 30 1
 10 1000 40 1
 11 1100 40 1
 12 1200 40 1
 13 1100 20 2
 14 1200 20 2
 15 1300 20 2
 16 1400 20 2
 17 850 30 2
 18 950 30 2
 19 1050 30 2
 20 1150 30 2
 21 1300 10 3
 22 1400 10 3
 23 1500 10 3
 24 1600 10 3


Transfection Conditions

Unless noted, transfections were carried out with the following conditions:
  • Cells were grown in Advanced RPMI + 4% FBS
  • Transfection mix was approx. 1ug pEGFP-C3 DNA (~4.7kb) per 240,000 cells
  • Transfection was carried out in 10ul tips, and cells grown in 24-well plate following electroporation
  • FL was measured at 24hr and 48hr following transfection


Transfection Results

Results were determined by FACS, measuring green fluorescent signal. The viable cells gate was set based on the forward/side scatter dot plot, capturing the viable cell population. Two FL gates were set based on the FL histogram looking at only viable cells: (1) M1, set so that <1.0% of the untransfected cells fell within the M1 gate, and then going all the way to the end of the FL plot (from the far right of the histogram, to the end). (2) M2, set arbitrarly at 10^2 FL units, to capture the more highly fluorescent cells. Numbers summarized below are % total viability, and then calculated % total cells that are viable and fall in the FL gate.


KMS12PE


          

LP1

Transfection done with approx. 1.8ug pEGFP-C3 DNA (~4.7kb) per 220,000 cells


RAJI



RPMI8226



U266

Transfection done with approx. 1.8ug pEGFP-C3 DNA (~4.7kb) per 225,000 cells



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