Electrotransfection of BHK-21 cells
• Dislodge cells with 1X trypisn/EDTA: 5-10 ml/150 cm2 flat. Incubate 37°C about 5 min.
Pipet cells up and down to obtain a uniform suspension.
• Mix with an equal volume of growth medium to stop trypsination.
• Spin 1.5-2 k to pellet cells
• Resuspend in 15ml ice-cold PBS. Spin to pellet (2X)
• Resuspend in 5ml cold PBS. (If it is known that 5ml is more volume than will ultimately be needed, resuspend the cells in an appropriate volume.)
• Count cells using a hemocytometer:
o 450ul PBS o 25ul cell resuspension o 25ul Evan’s blue (or Trypan blue) __________________________ 500ul Mix
• Apply about 25ul to hemocytometer. Count 4 squares having 16 sub squares/ square. Calculate average for the 4 squares.
o Calculate: Avg. #cells X 104 X dilution factor (20 here)
• Adjust cell volume with PBS to obtain 1X107 cells/ml
• Mix 400ul cells with 5-10ug RNA in sterile eppendorf tube. Keep on ice. (Need one 2mm gap cuvette/ electorporation) Electroporate 1 cell sample wo/RNA.
o Remove cuvette from ice immediately prior to electroporating. Warm by hand and pulse.
o Pulse conditions: 450V (450V setting) • 720 ohms (R10) • 100uF • Pulse length should be about 0.7msec.
• Pulse twice in succession.
• Add 15ml growth media and put into 1 T75 flask.
• Harvest virus (supernatant) at 3+ CPE. Should be between 24-48 hrs.